Recombinant protein carrying human papillomavirus epitopes inserted in an adenylate cyclase protein or fragment thereof. therapeutic uses thereof

ABSTRACT

The invention relates to a recombinant protein comprising one or several polypeptides bearing one or several epitopes of one or several HPV antigens, said polypeptides being inserted in the same or different permissive sites of an adenylate cyclase (CyaA) protein or of a fragment thereof, wherein said CyaA fragment retains the property of said adenylate cyclase protein to target Antigen Presenting Cells. It also concerns polynucleotides encoding the same. The recombinant protein or the polynucleotide can be used for the design of therapeutic means against HPV infection or against its malignant effects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/EP2005/003452, filed on Mar. 18, 2005, the content of which isincorporated herein by reference, and claims the priority of EuropeanPatent Application No. 04290741.0, filed on Mar. 18, 2004, the contentof both of which is incorporated herein by reference.

The present application relates to a recombinant protein carryingpapillomavirus epitopes inserted in an adenylate cyclase protein or afragment thereof.

Accordingly, the invention relates to recombinant proteins wherein theadenylate cyclase (CyaA) protein acts as a protein vector to elicit animmune response against epitopes derived from papillomavirus antigensespecially human papillomavirus antigens.

The invention especially relates to the use of the proteinaceous vectorthus obtained to deliver epitopes to eukaryotic cells, preferably tomammalian cells, and especially human cells.

The invention also concerns polynucleotides encoding the recombinantprotein of the invention, together with vectors containing saidpolynucleotides as well as host cells containing said polynucleotides orvectors.

The invention also relates to applications of the above recombinantprotein or polynucleotides for the treatment or prevention of humanpapillomavirus infection in a host as well as for the treatment orprevention of malignant effects resulting from infection, by humanpapillomaviruses, in a host, particularly in a mammalian host. In aparticular embodiment, the invention provides means useful for thedesign of compounds suitable for immunotherapy, especially immunotherapyagainst tumor specific antigens of papillomaviruses.

Among the numerous human papillomavirus (HPV) types, those designated ashigh-risk HPVs are linked with the development of epithelialmalignancies upon persistence of infection in the host (1). Cervicalcarcinoma, the second most widespread gynecological cancer worldwide(1), is associated (>99%) with the detection of mostly HPV16 and HPV18DNA (2). The oncogenic potential of these viruses is attributed to theproducts expressed by early genes, i.e., E6 and E7 genes whichexpression is detected throughout the replication cycle of the virus andis necessary for the onset and the maintenance of malignanttransformation. However, the high frequency of anogenital infection withthese high-risk oncogenic HPV types (3) contrasts with the lowproportion of individuals that will eventually develop HPV-associatedmalignancies, suggesting a control of high-risk HPV infections by immuneresponses. Several observations strengthened this statement such as thespontaneous regression of the majority of pre-malignant lesions (1), theinfiltration of regressing genital warts by CD4⁺ T cells and macrophages(1) as well as the higher number of infected subjects found inimmunosuppressed or immunodeficient patients (1). Furthermore, CD4⁺ andCD8⁺ T-cell responses against HPV16-E6 and/or E7 epitopes were detectedin the blood of patients diagnosed with HPV16-associated malignancies(4-8) as well as in the blood of healthy individuals (9, 10).Altogether, these considerations constituted a strong rationale for thedevelopment of immunotherapy targeting the E6 and/or E7 proteins ofHPV16.

Many vaccine strategies have been developed to prevent tumor growth ofHPV16-E6 and -E7-positive tumorigenic cell lines in C57BL/6 mice bygenerating immune responses to the H-2D^(b) HPV16-E7₄₉₋₅₇-restrictedepitope. These vaccination approaches have included plasmid DNA, viralor bacterial vectors, chimeric virus-like particles, synthetic peptidesand recombinant proteins (11). Unfortunately, those approaches yieldedmildly satisfying results in terms of clinical regression (3). Hence, itremains of interest to evaluate novel tools to target HPV-epitopes toimmune system for induction of cellular mediated responses.

There is thus a need for new vectors suitable for delivering epitopes ofHPV antigens to target cells, in conditions allowing the elicitation ofa humoral and/or cellular-mediated immune response in a host againstsaid antigens. The inventors have found that adenylate cyclase proteinmay be of interest in order to design such a vector. Variousobservations have been made, using adenylate cyclase protein ofBordetella pertussis, which led to the conclusion that this protein mayrepresent a suitable basis for the design of such efficient vector.

The adenylate cyclase (CyaA) of Bordetella pertussis has the capacity todeliver its catalytic domain into the cytosol of eukaryotic cells (12).Thus, CD4⁺ and CD8⁺ T cell epitopes inserted into the catalytic site ofCyaA are processed and presented by MHC class II and I molecules,respectively, at the surface of antigen-presenting cells (APC) (13).Furthermore, CyaA was recently demonstrated to bind specifically to theα_(M)β₂ integrin (CD11b/CD18) (14, WO 02/22169) and so to target theseT-cell epitopes to the CD11b⁺ dendritic cell subpopulation (15).Immunization of mice with a recombinant CyaA bearing appropriateepitopes led to the induction of strong CTL responses, full protectionagainst a lethal viral challenge and efficient prophylactic andtherapeutic antitumor immunity (16, 17). The adenylate cyclase (CyaA)protein has been characterized and disclosed for its preparation byrecombinant DNA technology especially in WO 93/21324 or WO 02/22169. InWO 02/22169, it has been described that fragments of CyaA encompassingresidues 373 to 1706 contain the structure essentially required forinteraction with the CD11b/CD18 receptor.

More specifically, it has been described later that the amino acidsequence extending from residue 1166 to amino acid residue 1281 comprisea determinant for interaction with the CD11b/CD18 receptor, and moreparticularly that amino sequence extending from residue 1208 to residue1243 are critical for the interaction of the toxin with CD11b/CD18 (EP03291486.3 and 45).

The inventors have now determined and evaluated conditions for theconstruction of a recombinant CyaA protein bearing, i.e., comprising,epitopes of HPV antigens, that can deliver said epitopes in targetcells, especially in Antigen Presenting Cells (APC), of a host,including hosts suffering from HPV infection and from its malignanttransformations.

Accordingly, the invention especially relates to a recombinant proteincomprising one or several polypeptides bearing one or several epitopesof one or several HPV antigens, said polypeptides being inserted in thesame or different permissive sites of an adenylate cyclase (CyaA)protein or of a fragment thereof wherein said CyaA fragment retains theproperty of said adenylate cyclase protein to target the target cells ofCyaA such as APC, especially CD11b/CD18 cells, such as dendritic cells.In a particular embodiment, this fragment also retains the property ofCyaA to allow translocation of the epitope inserted therein or of thepolypeptide containing said epitope into the cytosol of a target cell.Translocation of the epitope or polypeptide containing said epitope intothe cytosol of the target cell can be permitted if the fragment of CyaAretains the domain of the protein which permits translocation of itscatalytic domain.

The recombinant protein of the invention can be prepared having recourseto recombinant technology. It can also be obtained by synthesis,especially by chemical synthesis. Hence, the terms “recombinant protein”refers to the chimeric form of the protein.

The capacity of the recombinant protein to target CD11b/CD18 cells canbe assayed especially according to the methods disclosed in EP03291486.3 and (45) or in WO 02/22169. Furthermore, the capacity of therecombinant protein to translocate the epitope or polypeptide containingsaid epitope into the cytosol of target cell can be assayed by applyingthe method described in WO 02/22169.

In a particular embodiment, the fragment of CyaA can be constituted oftwo different portions of CyaA which are not naturally contiguous inCyaA. As an example, one may cite the catalytic domain of CyaA, i.e.,the 400 amino acid residues of the N-terminal part of CyaA and afragment comprising amino acid residues 1208 to 1243 required fortargeting of CD11b/CD18 Antigen Presenting Cells.

In the above definition, the expression “polypeptide” describes anyamino acid sequence, including amino sequences undergoingpost-translational modifications, especially amino acid sequence havingat least six amino acid residues, and including amino-acid sequenceshaving especially from 5 to 500 residues or from about 5 to about 100,or from about 5 to about 200 or from about 10 to about 50 residues, orfrom about 30 or about 50 to 200 residues, or from about 100 to about210 or from about 100 to about 200 residues providing said amino acidsequence comprises at least one epitope, i.e., an amino acid sequenceagainst which an immune response may be obtained after its delivery to atarget cell, advantageously in a host, especially in a mammal host.Polypeptides according to this definition can thus be restricted toepitopes, even to a unique epitope or can comprise several different oridentical epitopes or can also encompass full-length antigens from apathogen, i.e., from human papillomavirus. Epitopes within the presentinvention encompass amino acid sequences which are involved in humoralimmune response and/or cell-mediated immune response, especially in Tcell immune response. Accordingly, epitopes in the polypeptides of therecombinant molecules of the invention include those which are processedby APC (Antigen Presenting Cells) in a host, especially those recognizedin association with class I MHC (Major Histocompatibility Complex)molecules such as epitopes which target cells are CD8⁺ T lymphocytes orepitopes recognized in association with class II MHC molecules such asthose which target cells are CD4⁺ T lymphocytes cells.

In a particular embodiment, the polypeptide bearing epitopes comprisesseveral epitopes derived from different antigens, especially from onetype of antigen of different HPV strains or from several types ofantigens of different HPV strains. Hence, the polypeptide derived fromHPV antigens can be multivalent, especially bivalent or trivalent, i.e.,enabling an immune response against several antigens.

According to the present invention, HPV antigens from which thepolypeptides bearing one or several epitopes can be designed, arepreferably those derived from proteins especially involved in the onsetand/or maintenance of malignant effects following HPV infection andencompass so-called tumor antigens, i.e., antigens associated with tumordevelopment related to HPV infection, that can elicit an immune responsein a host and react specifically with antibodies or T-cells in a host.

The polypeptides bearing epitopes according to the invention may bederived from native or mature antigens of HPV including by using thewhole antigen or including by selecting fragments, especially antigenicfragments, especially epitopes of said antigens, rather than the wholeprotein or by modifying said antigen or its selected antigenic parts orepitopes, especially in order to improve their capacity to induce orelicit an immune response in a host when combined with CyaA protein inthe recombinant molecule. Accordingly, to illustrate the variouspossible modifications of such epitopes, these polypeptides encompassepitopes which are flanked by naturally or non-naturally flankingsequences of the antigen from which they are derived, and also encompassepitopes or amino acid sequences containing epitopes which have beenchemically modified in order to improve their immune properties. Thesemodifications can be advantageous to improve the efficiency of theobtained polypeptides in the association with CyaA protein.

Some particular modifications are disclosed as examples hereafter,including modifications encompassing changes in the charge of thepolypeptides, especially by insertion of additional positively-chargedamino acid residues.

Accordingly, the polypeptide of the invention also encompassessemi-synthetic or synthetic polypeptide.

According to a particular embodiment, the polypeptides derived from HPVantigens contain, each or together, from about 5 to about 500, or fromabout 5 to about 100, or from about 5 to about 200, for example fromabout 10 to about 50 amino acid residues or from about 30 or about 50 toabout 200 amino acid residues or from about 100 to about 210 or fromabout 100 to about 200 amino acid residues.

The polypeptide(s) is (are) chosen especially to enable elicitation ofan antigen-specific response when recombined in the recombinant proteinof the invention.

The recombinant protein of the invention can especially be designed tocomprise a polypeptide or several polypeptides consisting in a disruptednative HPV antigen, wherein said disruption consists of a deletion ofone or several amino acid residues in an acidic region of said HPVantigen, and/or an insertion of at least two polypeptide fragments ofsaid HPV antigen in at least two permissive sites of the adenylatecyclase.

A particular disruption encompassed within this definition, is obtainedby insertion of at least two fragments of the native HPV antigen, in atleast two permissive sites of the adenylate cyclase, wherein these atleast two fragments are reversed with respect to their natural locationin the native antigen, i.e., the fragment which in the native antigen ismore N-terminal becomes C-terminal when inserted in the CyaA protein orfragment thereof, and vice-versa.

It has been observed that the inversion of amino-terminal andcarboxy-terminal fragments can be more effective in inducing strong andlong-lasting protective immunity, especially in cancer immunotherapy, asillustrated with E7_(Δ) fragments (i.e., fragments of the E7 antigen).

According to the invention, adenylate cyclase (CyaA) is used as afull-length protein or as a fragment thereof, as disclosed above.

Advantageously, the CyaA protein or a fragment thereof is a protein or afragment thereof, which is the result of the co-expression in a cell,especially in a recombinant cell, of both cyaA and cyaC genes. It hasbeen indeed shown that in order to have invasive properties for targetcells, CyaA has to undergo post-translational modifications which areenabled by the expression of both cyaA and cyaC genes (WO 93/21324).

In a particular embodiment of the invention, fragments of the CyaAprotein are fragments having at least about 30 amino acid residues andcan have up to about 1300, in particular to about 500 amino acidresidues, preferably from about 50 to about 150 amino acid residues;said fragments comprise, in a particular embodiment, amino acid residues1166 to 1281 of CyaA or amino acid residues 1208 to 1243 of CyaA proteinfor interaction with CD11b/CD18 target cells. A particular fragment thusencompasses all or part of the C-terminal part of the native proteinwhich part is responsible for the binding of the protein to target cellmembrane and/or CD11b/CD18 receptor, and for the subsequent delivery ofthe epitope(s) contained in the polypeptide(s) into the cell cytosol(12). A particular fragment of CyaA protein according to the inventioncontains amino acid residues 372 to 1706 of CyaA protein. Anotherparticular fragment is one which corresponds to the CyaA protein whereinamino acid residues 225 to 234 have been deleted, thus providing a CyaAfragment containing residues 1 to 224 and 235 to 1706.

In a particular embodiment of the invention, the adenylate cyclaseprotein is a bacterial protein. In a preferred embodiment, CyaA proteinis derived from a Bordetella species.

Among Bordetella species of interest, according to the invention, one ofthem is Bordetella pertussis. Other Bordetella strains of interest arethose of Bordetella parapertussis or Bordetella bronchiseptica. Thesequences of CyaA protein of B. parapertussis, has been disclosedespecially under accession number NC 002928.3 (as a sequence of 1740amino acids) and in Parkhill J. et al (Nat. Genet. DOI, 10 (2003) andfor B. bronchiseptica in Betsou F. et al (Gene 1995, Aug. 30; 162(1):165-6).

Bordetella pertussis is the causative agent of whooping cough andsecretes among others several toxins including the well-known pertussistoxin (PT) and the adenylate cyclase toxin (CyaA), which is a criticalvirulence factor of the bacterium and is one of the antigens protectiveagainst B. pertussis infection.

The adenylate cyclase protein of Bordetella pertussis is a toxin whichhas been described as a bifunctional protein of 1706 residues,comprising a N-terminal catalytic domain of 400 amino acid residues anda C-terminal part of 1306 residues which is responsible for the bindingof the toxin to target cell membrane and subsequent delivery of thecatalytic moiety into the cell cytosol (12).

The CyaA protein is synthesized as an inactive protoxin which isconverted into an active toxin by post translational palmitoyation oftwo internal lysine residues (lysins 860 and 983). This posttranslational modification requires the expression with the cyaA gene ofan accessory gene, i.e., cyaC which is located nearby cyaA on B.pertussis chromosome.

The cyaA of Bordetella pertussis has been described as an amino acidsequence and a nucleotide sequence by Glaser, P. et al, 1988, MolecularMicrobiology 2(1), 19-30. Accordingly, when amino acid residues orsequences or nucleotides or nucleotide sequences of the CyaA protein ofB. pertussis, are cited in the present invention their positions aregiven with respect to the sequences disclosed in said publication ofGlasser et al. 1988.

In the recombinant protein according to the invention, the polypeptidesbearing one or several epitopes of one or several HPV antigens, areinserted in one or several permissive sites of the CyaA protein.

For the present invention, a “permissive site” is a site of the sequenceof the CyaA protein where a polypeptide can be inserted withoutsubstantially affecting the functional properties of the CyaA proteinespecially without substantially affecting the targeting of cells,particularly targeting of APC by CyaA, including without substantiallyaffecting the specific binding to the CD11b-CD18 receptor andadvantageously without substantially affecting the domains of theprotein involved in the process of translocation of the epitope(s) intoa target cell.

Permissive sites of the Bordetella pertussis adenylate cyclase allowingtranslocation of CyaA catalytic domain and hence translocation ofepitopes inserted into such permissive sites include, but are notlimited to, residues 137-138 (Val-Ala), residues 224-225 (Arg-Ala),residues 228-229 (Glu-Ala), residues 235-236 (Arg-Glu), and residues317-318 (Ser-Ala) ((44) Sebo et al., 1995). The following additionalpermissive sites are also included in embodiments of the invention:residues 107-108 (Gly-His), residues 132-133 (Met-Ala), residues 232-233(Gly-Leu), and 335-336 (Gly-Gln) and 336-337. (43)

For other Bordetella species corresponding permissive sites can bedefined by comparison of sequences and determination of correspondingresidues.

According to another embodiment, the polypeptide can also oralternatively be inserted at one and/or other extremities of CyaAprotein or its fragment.

Particular fragments of CyaA proteins for use for the purpose of theinvention are those comprising up to 1300 amino acids or from about 30to about 500 amino acid residues, advantageously about 50 to about 150amino acid residues in particular such fragments encompassing amino acidresidues 1166 to 1281 of the native CyaA protein, advantageously 1208 to1243 of native CyaA protein.

Thus, according to the invention, the “insertion” of a polypeptide inthe CyaA protein to provide a so-called recombinant protein alsoreferred to as a “hybrid protein”, encompasses genetic insertionespecially by available DNA technology. Alternatively, “insertion” alsoencompasses non genetic insertion, including chemical insertion forinstance covalent coupling carried out at one extremity of the CyaA orfragment thereof, or non covalent coupling. Non-genetic insertion canespecially be of interest when the polypeptide to be inserted issynthetic or semi-synthetic. Methods for coupling a drug to apolypeptide are well known in the Art and comprise for example disulfidelinkage by using N-pyridyl sulfonyl-activated sulfhydryl.

In particular, it is possible to graft molecules especially comprisingpolypeptides of the invention to CyaA by a chemical linkage or bygenetic insertion for in vivo targeting to target cells of Cya, such asACP, for example CD11b/CD18 cells and particularly to the cytosol ofsaid cells. Indeed, when coupling a molecule corresponding to a givenCD8⁺ T-cell epitope to the catalytic domain of detoxified CyaA, eitherby means of a disulfide bond or by genetic insertion, it has been foundthat the engineered molecule can elicit in vivo specific CTL response,thereby showing that said CD8⁺ T-cell epitope is translocated into thecytosol of CD11b-expressing cells.

In a specific embodiment, the recombinant adenylcyclase used for themanufacturing of proteinaceous vector is a CyaA or fragment thereofespecially modified by insertion of cysteine residues containing one ormore molecule(s), especially comprising polypeptides of the invention,chemically coupled by means of a disulfide bond to genetically insertedcysteine residue(s) located within the catalytic domain of saidadenylcyclase.

Indeed, multiple molecules especially comprising polypeptides of theinvention, can be chemically coupled to the adenylcyclase by means of adisulfide bond to different cysteine residues located at differentpermissive sites within the catalytic domain.

With a view to propose a recombinant protein suitable for the design ofproducts having the capacity to elicit an immune response, especially acell-mediated immune response in a host, and in particular in order todesign such products capable of eliciting an immune response against themalignant effects observed in a host infected with HPV, the inventorshave proposed to derive polypeptides bearing epitopes from highlyoncogenic HPV strains and especially from antigens from strains selectedamong HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52 or HPV58.

Among these strains, HPV 18 and HPV 16 are of particular interest. HPV16 is especially a particular target for treatment of a host infectedwith HPV, because of its association with the development of cervicalcancer in mammal host especially in human.

Starting from these HPV strains, the inventors propose to derivepolypeptides bearing epitopes from antigens selected among L1, L2, E1,E2, E4 and E5 proteins.

Alternatively or in combination, the inventors also propose to derivesuch polypeptides bearing epitopes from E6 or E7 proteins of HPV.

In a particular embodiment of the invention, E6 or E7 proteins of HPV16or E6 or E7 proteins from HPV18 are used for the design of polypeptidesbearing epitopes.

A particular HPV protein which can be referred to for the design of apolypeptide derived from HPV antigens is the E7 protein of HPV,especially of HPV16 or of HPV18. According to an embodiment of theinvention, the polypeptide is derived from several E7 proteins ofdifferent HPV strains, especially of HPV16 and HPV18. For example, thepolypeptide is the full length E7 protein of HPV 16 and HPV18 or one orseveral fragments of each of the E7 protein of HPV16 or HPV18, includingmultimers, especially dimers of said fragments.

These proteins of HPV and their amino acid and nucleotide sequences havebeen disclosed in Seedorf, K. et al (Human papillomavirus type 16 DNAsequence. Virology, 145: 181-185, 1985) for HPV16, Cole S. T., Danos O.(Nucleotide sequence and comparative analysis of the humanpapillomavirus type 18 genome. Phylogeny of papillomaviruses andrepeated structure of the E6 and E7 gene products. J. Mol. Biol. 193:599-606 (1987)) or in Fernando G J. et al (T-helper epitopes of the E7transforming protein of cervical cancer associated human papillomavirustype 18 (HPV18) Virus Res. 1995 Apr. 36(1): 1-13).

The E6 and E7 proteins are oncoproteins expressed especially by HPV16 orHPV18 throughout the replicative cycle of the virus and they have shownto be necessary for the onset and maintenance of malignanttransformation of host cells, following infection with HPV strain.Therefore, both these tumors specific antigens are considered aspotential targets for adoptive CTL-mediated immunotherapy.

According to a particular embodiment of the invention, the recombinantprotein comprises multiple polypeptides, each of them bearing one orseveral epitopes of one or several HPV antigens.

For example, such multiple polypeptides can be derived from E6 and E7proteins of one HPV strain, especially from HPV16 or HPV18. According toanother example, these multiple polypeptides can encompass epitopesderived from E6 or E7 proteins, from both HPV16 and HPV18.

Multiple polypeptides can also consist of different epitopes bearingfragments of one protein, for example of an E7 or E6 protein, which areinserted in different permissive sites of the CyaA protein of interest.

Another particular recombinant protein according to the abovedefinitions is a protein CyaA recombinant wherein the multiplepolypeptides bearing epitopes encompass a fragment comprising residues 1to 29 or a fragment consisting of residues 1 to 29 or a fragmentcomprising residues 42 to 98 or a fragment consisting of residues 42 to98 of E7 protein of HPV16, or multiple polypeptides comprising orconsisting of both fragments, inserted in different permissive sites ofthe CyaA protein.

Another recombinant protein according to the invention is a proteinwherein the multiple polypeptides encompass a fragment having amino acidsequence RAHYNIVTF (SEQ ID NO: 1) (E7₄₉₋₅₇) and/orGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 2) (E7₄₃₋₇₇).

It has been observed that the number of amino acid residues of thepolypeptides inserted in permissive sites of the CyaA protein is suchthat it allows for polypeptides consisting of full-length antigens,especially of full-length E6 or E7 proteins of HPV to be inserted inCyaA protein or fragments thereof.

According to a particular embodiment of the invention, the polypeptideincluded in the recombinant CyaA is the E7 protein, especially the E7protein of HPV16, inserted between codons 224 and 235 of CyaA or betweencodons 319 and 320 of CyaA.

In another embodiment, the recombinant protein of the inventioncomprises multiple polypeptides, some of which being polypeptidesbearing an epitope or several epitopes of one or several HPV, and otherpolypeptides bearing epitopes of other pathogens.

In another particular embodiment, the recombinant protein of theinvention further comprises one or several epitopes originating from adifferent pathogen agent. Association of epitopes originating fromChlamydia or from HIV retrovirus or HPV, HBV, HCV, adenoviruses EBV,herpes virus, HTLV.1 virus and CMV, with epitopes originating from HPVmay especially be of interest.

According to another particular embodiment of the invention, thepolypeptides bearing epitopes have been modified with respect to theirnative amino acid sequence, for example in order to decrease the numberof negatively charged amino acid residues within the sequence. Such amodification can be obtained by removing some of these negativelycharged amino acid residues or also by adding some positively chargedamino acid residues, especially as flanking residues of the epitopes.Polypeptides thus comprising less negatively charged residues mightfavour the translocation of the catalytic domain of CyaA protein in thecytosol of target cells.

The polypeptides bearing epitopes can also be designed in such a waythat they are unfolded when they are inserted in CyaA, which improveefficiency of the internalization of the recombinant CyaA protein intothe target cells. Such unfolding in polypeptides which undergo foldingas a consequence of their amino acid content, can be obtained forinstance, by removing or substituting cystein residues in order to avoidformation of disulfide bonds that may be involved in folding ofpolypeptides. In some cases, it is possible to prevent folding of thepolypeptides by preparing them in the presence of reducing agents toenable avoiding in vivo refolding.

In a particular embodiment, the polypeptides bearing epitopes can becryptic epitopes.

In a particular aspect of the invention, the inventors have indeeddetermined that the chimeric protein constructs, made of the recombinantproteins which comprise (i) an adenylate cyclase (CyaA) or a fragmentthereof according to the definitions disclosed above and (ii) apolypeptide bearing one or several antigenic fragments of one or severalantigens, enable cryptic epitopes of said antigens to become immunogenicas a result of their presentation in the recombinant construct.Especially, said chimeric constructs involving CyaA or a fragmentthereof as defined in the present invention and polypeptides derivedfrom antigens of interest for especially therapeutic, includingvaccinating, purposes can comprise cryptic epitopes of the antigen whichare allowed to become immunogenic and in particular to raise a T-cellresponse in a host, especially a CTL response.

The invention thus also relates to a recombinant protein comprising oneor several polypeptides bearing one or several epitopes of one orseveral antigens, said polypeptide(s) being inserted in the same or indifferent permissive sites of an adenylate cyclase (CyaA) protein or ofa fragment thereof, said CyaA fragment retaining the property of saidadenylate cyclase protein to target Antigen Presenting Cells, wherein atlease one of said epitope(s) is a subdominant cryptic T-cell epitope andwherein said recombinant protein is capable of eliciting anantigen-specific response against said polypeptide(s).

Especially, the cryptic epitopes are contained within an HPV antigen, inparticular HPV16 and/or HPV18 antigens, especially an E7 antigen.

The recombinant protein thus defined especially comprises a peptidederived from HPV18 E7 protein, i.e., having amino acid sequenceIDGVNHQHL (SEQ ID NO: 3).

According to a particular embodiment the cryptic epitope can be modifiedfor example can have substitutions in the two first positions, and forinstance can have sequence ASGVNHQHL (SEQ ID NO: 4).

The invention especially concerns peptides IDGVNHQHL (SEQ ID NO: 3).

The invention also concerns peptides having substitutions in thissequence, especially at positions 1 and/or 2, in particular peptideshaving sequence ASGVNHQHL (SEQ ID NO: 4).

The invention also comprises variants of said peptides, to the extentthat they have immunogenic properties, especially capable of eliciting aT-cell, in particular a CTL response.

Advantageously, in order to prepare the recombinant protein of theinvention, the enzymatic activity of the CyaA protein, i.e., its abilityto convert ATP into cAMP, has been inactivated. Such inactivation may beobtained as a result of genetic inactivation. As an example, geneticinactivation can be obtained as a result of introduction of a dipeptidein a site of the amino acid sequence of CyaA which is part of thecatalytic site (for example between 188 and 189). Such inactivated CyaAproteins are illustrated in the following examples.

The recombinant protein of the invention is advantageously capable ofeliciting a cell-mediated immunoresponse. It includes CTL and Th,especially Th1 response, including CD4⁺ T cell response and/or CD8⁺ Tcell response.

The ability of the recombinant protein to elicit this cell-mediatedimmune response has especially been shown to be sufficient to preventtumor growth in vivo or even to enable tumor regression in an animal.

The invention also relates to a polynucleotide which encodes arecombinant protein as defined above.

A polynucleotide of the invention can be inserted in an expressionvector to provide a recombinant expression vector suitable forexpression of the recombinant protein of the invention. Such expressionvectors include plasmids, cosmids, phagemids, viral vectors.

A recombinant vector can be one which is suitable for expression inprokaryotic cells, especially in bacteria or can be an expression vectorsuitable for expression in eukaryotic cells especially in mammaliancells, and advantageously in human cells.

The invention especially relates to vectors consisting of plasmidsencoding a recombinant protein according to the invention such as:pTRACE5-HPV16E7_(Full) (also designated CyaAE5-HPV16E7_(FULL)),deposited at the CNCM (Paris, Francc) COLLECTION NATIONALE DE CULTURESDE MICROORGANISMES (CNCM). 25 rue du Docteur Roux. F-75724 PARIS Cedex15. FRANCE on Mar. 18, 2004 under number CNCM 1-3191;pTRACE5-HPV16E7_(Δ30-42), (also designated CyaAE5-HPV16E7_(Δ30-42)),deposited at the CNCM (Paris, Francc) COLLECTION NATIONALE DE CULTURESDE MICROORGANISMES (CNCM). 25 rue du Docteur Roux. F-75724 PARIS Cedex15. FRANCE on Mar. 18, 2004 under number CNCM 1-3190, or constructpTRACE5-HPV16E7₄₉₋₅₇.

The invention also comprises a host cell, especially prokaryotic cells,or eukaryotic cells, for example mammalian cells, including human cells,transformed with a polynucleotide or a vector according to theinvention.

The invention especially relates to the host cells deposited at the CNCMunder accession N^(o) CNCM I-3190 and accession N^(o) CNCM I-3191.

The invention also concerns an immunogenic composition which comprisesas an active principle, a recombinant protein as defined above or apolynucleotide or an expression vector as defined above. Said activeprinciple of the immunogenic composition can be formulated inassociation with a physiologically acceptable, vehicle, excipient,carrier or diluent or a combination thereof, suitable for administrationto a host.

An immunogenic composition is advantageously designed to induce acell-mediated immune response, in particular a T-cell mediated immuneresponse, in a mammal host. Preferably, it is capable of inducing acell-mediated cytolytic immune response CTL especially CD8+.

Another immunogenic composition according to the invention is one whichcan induce a humoral immune response.

In order to improve the capacity of the immune composition of theinvention to induce an immune response, it may be interesting to combinethe active principle with an adjuvant and/or a surfactant and/orimmunomodulatory substances (such as cytokines or chemokines).

Adjuvant include, for example, liposomes, oily phases, such as Freundtype adjuvents, generally used in the form of an emulsion with anaqueous phase or can comprise water-insoluble inorganic salts, such asaluminium hydroxide, zinc sulphate, colloidal iron hydroxide, calciumphosphate or calcium chloride.

The immunogenic composition according to the invention is advantageouslyused to induce an immune response in a host, either by priming and/or byboosting said response, especially for immunotherapy. Especially, animmunogenic composition of the invention can be of interest forprevention on the onset or maintenance of malignant transformation dueto HPV infection in a host or for treatment of a patient suffering frommalignant transformation due to HPV infection, especially HPV-16 orHPV-18 infection.

Such an immunotherapeutic composition may be of particular interest fortherapy of uncontrolled cell proliferation in a host resulting in atumorigenic state, especially for cancer immunotherapy in particular forcervical cancer immunotherapy associated with HPV infection. Ittherefore provides means for the design of therapeutic vaccinesespecially suitable for the treatment of malignant states due tooncovirus infections, including tumor states.

When used in the present invention, the expressions “treatment” or“therapeutic treatment” encompass the effects of the compounds disclosedin the present application, which result in a beneficial effect for thepatient undergoing the treatment, said effects being either observed ata cellular level or at a clinical level, including encompassing, as aresult of the treatment, an improvement of the condition of the patientor a remission state, or a recovery of a health state. When themalignant state treated is uncontrolled cell proliferation or tumordevelopment or persistence, the beneficial effect can comprise thestabilization or preferably the prevention, stopping or reversal ofuncontrolled proliferation or the regression of the tumor.

A composition intended for the treatment of a malignant state asdescribed above can advantageously comprise a dose of active principlewhich can amount to from about 1 to about 1000 μg of recombinantprotein, preferably from about 10 to about 500 μg of a recombinantprotein. When the composition comprises as active principle arecombinant protein of the invention the dose can comprise from about0.05 to about 10 μg of recombinant protein, preferably from about 0.1 toabout 1 μg of protein.

Depending on the state to be treated, the composition can beadministered locally at the level of the lesion, once or several times,for example at regular intervals of several days, for example, for 5 to10 days. It can also be administered systemically.

The invention also relates to a vaccine composition, especially acomposition formulated for administration to a mammal host, preferablyto a human, comprising a recombinant protein according to the abovedefinition or a polynucleotide as defined hereabove or a vectorcontaining such polynucleotide, preferably in a human host, and ifappropriate a pharmaceutically acceptable vehicle, for eliciting animmune response, including a cell-mediated immune response, and/or ahumoral response.

The invention relates also to a drug composition comprising arecombinant protein or a polynucleotide or a vector of the invention,and a pharmaceutically acceptable vehicle, for preventing or treatingHPV infections.

According to another embodiment, a drug composition comprises arecombinant protein according or a polynucleotide or a vector of theinvention, with a pharmaceutically acceptable vehicle, for prevention ortreatment of the onset or maintenance of malignant transformation due toHPV infection in a host.

A drug composition comprising a recombinant protein or a polynucleotide,or a vector and a pharmaceutically acceptable vehicle, for cancerimmunotherapy.

The invention also concerns the use in a patient of recombinant proteinscomprising a bacterial protein especially a bacterial toxin (preferablyin their toxoid form) or a fragment thereof, suitable to be used as avector to elicit an immune response, i.e., a humoral and/or acell-mediated immune response in a host which protein or fragmentthereof is modified by insertion of one or several epitopes of one orseveral antigens of one or several oncoviruses for the treatment of oneoncovirus infection. Such a recombinant protein is proposed inparticular for the treatment of malignant effects, especially tumorscaused by infection by such oncovirus.

Examples of bacterial proteins suitable as vectors to carry epitopes ofantigens of oncoviruses are OmpA from klebsiella or the following toxinsShiga toxin including its β subunit (Haicher N. et al J. Immunol. 2000,165: 3301-8) Anthrax toxin (Goletz T J et al, PNAS USA 1997, 94:12059-64), Diphtheria Toxin (Stenmark H. et al, J. Cell. Biol. 1991,113: 1028-32) or Pseudomonas Exotoxin A (Donnelly J J. et al, PNAS USA1993, 90: 9530-4). Oncoviruses which antigens can provide epitopes forpreparing polypeptides for insertion in the bacterial protein includesHPV, HBV, HCV, adenoviruses EBV, herpes virus, HTLV.1 virus and CMV.

The description which is provided hereabove for the CyaA recombinantprotein, uses as an active principal, could be adapted for otherbacterial proteins and oncoviruses antigens.

The invention also relates to a kit for the diagnosis of an infectionwith an HPV or for immunomonitoring such infection, which comprises arecombinant protein, a polynucleotide or an expression vector accordingto the invention.

The invention also concerns the use of the above recombinant protein,polynucleotide or vector of the invention, for the treatment or for theprevention of HPV infection in a patient.

The invention also concerns the use of the above recombinant protein,polynucleotide or vector of the invention, for immunotherapy against theonset or maintenance of malignant transformation due to HPV infection ina patient.

The invention also relates to a method for the in vitro diagnosis or forthe immunomonitoring of an infection with HPV, comprising:

-   -   exposing T cells obtained from a mammal, especially from a human        patient, to a recombinant protein of the invention,    -   detecting a modification in the activation of T cells.

In a particular embodiment, the recombinant protein can be used forprevention of infection by HPV or for the treatment of hosts sufferingfrom infection by HPV, including hosts harbouring tumor due to suchinfection.

The invention also relates to a process for the screening of unknown orsub-dominant cryptic T-cell epitopes in a polypeptide contained in achimeric CyaA-polypeptide protein, wherein CyaA is the adenylate cyclaseor a fragment thereof as disclosed above, which comprises:

-   -   administering said chimeric protein to an animal host,    -   determining the T-cell response of said host, especially the CTL        response.

The invention especially relates to a process for screening unknown orsub-dominant or cryptic T-cell epitopes (especially CD8⁺ T-cellepitopes) in the polypeptide(s) of HPV-antigen(s) contained in arecombinant protein defined within the invention, which processcomprises:

-   -   administering said recombinant protein to an animal host        (non-human)    -   determining the T-cell response of said host, especially the CTL        response.

Further features characterizing the invention are disclosed andillustrated in the examples which follow and in the figures.

FIGURE LEGENDS

FIG. 1. Construction and purification of HPV16-E7 recombinant CyaAs.

(A) Schematic map of pTRACE5 in which relevant restriction sites andinserted sequences are indicated. (B) Schematic representation of CyaAshowing the site of insertion of the dipeptide LQ to inhibit itsenzymatic activity. Positions of the HPV16-E7 protein inserts are alsoshown. The HPV16-E7H-2b restricted epitope is underlined (SEQ ID NOS23-26 are disclosed respectively in order of appearance). (C) SDS-Pageanalysis of the HPV16-E7 recombinant CyaAs. Five micrograms of thepurified proteins were separated on a 4-15% SDS polyacrylamide gel andstained by Coomassie blue. Lane 1: wild-type CyaA; lane 2:CyaA-E7₄₉₋₅₇;lane 3: CyaA-E7_(Full); lane 4:CyaA-E7_(Δ30-42). (D) Western blotanalysis of the HPV16-E7 recombinant CyaAs. Following SDS-PAGE, purifiedproteins were electro-transferred onto a nitrocellulose membrane thatwas subsequently probed with a mouse monoclonal anti HPV16-E7 antibody.Lane 1, 2: wild-type CyaA (2 and 0.4 μg, respectively); lane 3, 4 and5:CyaA-E7₄₉₋₅₇,CyaA-E7_(Full) and CyaA-E7_(Δ30-42), respectively, 0.4 μgof each protein.

FIG. 2. Induction of T cell responses by recombinant HPV16-E7 CyaAs.

(A) C57BL/6 (a, b, c), TAP1^(−/−) (d), MHC class II^(−/−) (e) andCD40^(−/−) (f) mice were immunized i.v. on day 0 with 50 μg ofCyaA-E7₄₉₋₅₇ (a), CyaA-E7_(Full); (b), or CyaA-E7_(Δ30-42). (c, d, e andf). Seven days later, the animals were sacrificed, the splenocytes wererestimulated in vitro for 5 days with 1 μg/ml of the HPV16-E7₄₃₋₇₇peptide in the presence of irradiated syngeneic splenocytes, and used aseffectors against TC-1 target cells (plain squares) or EL4 (opensquares). Splenocytes from mice treated with CyaAE5-cysOVA carrying thenon relevant epitope OVA₂₅₇₋₂₆₄ and restimulated in vitro for 5 dayswith 1 μg/ml of the HPV16-E7₄₃₋₇₇ peptide in the presence of irradiatedsyngeneic splenocytes are also represented (a, plain triangles). Targetlysis was evaluated by ⁵¹Cr release. The data represent the medianpercentage of the specific lysis values (n=number of animals isindicated on each graph) as well as the interquartile ranges. (B)Detection of HPV16-E7-specific IFN-γ-producing cells after immunizationwith the recombinant HPV16-E7 CyaAs. C57BL/6 mice were immunized as in Awith CyaAE5-cysOVA (circles), CyaA-E7₄₉₋₅₇ (squares), CyaA-E7_(Full)(triangles), or CyaA-E7_(Δ30-42) (diamonds). Seven days later, spleencells isolated from immunized mice were cultured in vitro for 36 hwithout stimulation (i.e., no peptide, open symbols) or with 1 μg/ml ofthe E7₄₉₋₅₇ peptide (plain symbols) in the presence of syngeneicirradiated splenocytes. The data are expressed as the number of SFC perspleen and the result of individual mice of three independentexperiments are represented for each group. Horizontal bars representthe median response of each group.

FIG. 3. Recombinant HPV16-E7 CyaAs induce a HPV16 E7-specific Th1response.

(A) C57BL/6 mice were either left untreated (squares) or were primedi.v. with 50 μg of CyaAE5-cysOVA (circles), CyaA-E7₄₉₋₅₇ (triangles),CyaA-E7_(Full) (inverted triangles), or CyaA-E7_(Δ30-42) (diamonds).Seven days later, spleen cells were stimulated in vitro with 10 μg/ml ofthe His-Tag-HPV16-E7 protein, and the supernatants were tested for IFN-γcontent 72 hours later. Results of individual mice of 4 independentexperiments are represented and expressed as the concentration of IFN-γreleased in the supernatant from duplicate wells. Backgrounds obtainedwith non restimulated splenocytes are subtracted. Inset: in vitrostimulation with 1 μg/ml E7₄₃₋₇₇ peptide. Horizontal bars represent themedian response of each group. (B) Same as in (A) except thatsupernatants were tested for IL-5 content. Results of individual mice of2 independent experiments are represented and expressed as theconcentration of IL-5 released in the supernatant from duplicate wells.

FIG. 4. Therapeutic vaccination with recombinant HPV16-E7 CyaAseradicates established tumors.

(Exp. A) C57BL/6 mice were grafted on day 0 with 5×10⁴ TC-1 tumor cells.On day 10, mice were treated with one i.v. injection of CyaA-E7₄₉₋₅₇(C), CyaA-E7_(Full) (D), or CyaA-E7_(Δ30-42) (E). Mice left untreated(A) or injected with a CyaAE5-cysOVA (B) were taken along as controls.Mice were killed when the tumor size reached 1000 mm³ or whenever thesanitary status of the animals commanded (necrosed tumor, rapid weightloss>20%) so as to avoid unnecessary suffering. Two mice treated withCyaA-E7_(Full) that lately developed progressive tumors (*) weresacrificed for further investigation (see FIG. 6).

(Exp. B) Same as in (Exp. A) for the experimental setting. Therapeuticvaccination was performed in the ear dermis on days +10 and +17 with 10μg of CyaAE5-CysOVA (a, solid lines) or 10 μg of CyaA-E7_(Δ30-42) (b).Each curve represents the tumor growth in a single animal. Two untreatedanimals were included (a, dashed lines). In the top right of eachquadrant (a, b) is indicated the number of sacrificed animals vs thetotal number of animals included. Survival curves of these mice areshown (c). Untreated (open triangles), mock-treated with CyaAE5-CysOVA(closed triangles), treated with CyaA-E7_(Δ30-42) (circles).

FIG. 5. Therapeutic vaccination with recombinant HPV16-E7 CyaAs resultsin prolonged survival. Therapeutic vaccination was carried out asdescribed in FIG. 4. Upon injection with TC-1 tumor cells, mice (5 to 10per group) were immunized with HPV16-E7 recombinant CyaAs at day +1, +5or +10 as indicated on the graphs. Mice were left untreated (plainsquares, solid line), mock-treated with CyaAE5-cysOVA (open squares,dashed line), or treated with CyaA-E7₄₉₋₅₇ (open triangles),CyaA-E7_(Full) (open circles), or CyaA-E7_(Δ30-42) (open diamonds). Itis noted that in the day +5 therapeutic experiment, the survival curvesof animals treated with CyaA-E7_(Full) and CyaA-E7_(Δ30-42) arecompletely superimposed. In every case, the survival of recombinantHPV16-E7 CyaAs-treated animals is significantly increased as compared tothat of untreated or mock-treated mice (p<0.05).

FIG. 6. TC-1 tumor cells explanted from lately growing tumors lose theexpression of H-2D^(b) molecule.

In tumor rejection experiments, some animals vaccinated withCyaA-E7_(Full) grew tumors lately in the time course of the experiments(FIG. 4, *). Two animals were sacrificed to explant the tumors. Thesetumor cells, TC-1 A1 and A2 as well as native TC-1 cells were analyzedby FACS® for the level of expression of the H-2D^(b) molecule (boldline). The medians of fluorescence intensities (MedFi) are indicated.Results obtained with isotype control are shown (gray shaded).

FIG. 7. Persistence of HPV16-E7₄₉₋₅₇ specific CD8⁺ T-cells in micetreated with recombinant HPV16-E7 CyaAs.

C57BL/6 mice immunized with CyaA-E7₄₉₋₅₇ (A), CyaA-E7_(Full) (B), orCyaA-E7_(Δ30-42) (C) and surviving from TC-1 grafts in the therapeuticset of experiments were sacrificed and splenocytes were restimulated invitro for 5 days with 1 μg/ml of the HPV16-E7₄₃₋₇₇ peptide in thepresence of irradiated syngeneic splenocytes. Target lysis (TC-1, plainsquares; EL4, open squares) was evaluated by ⁵¹Cr release. The datarepresent the median percentage of the specific lysis values (n=6 foreach group) as well as the interquartile range. The number of respondinganimals, as determined by a specific lysis≧20% at the maximum effectorto target ratio, is indicated in the upper right part of the quadrants.

FIG. 8. Long term protection against TC-1 tumor growth induced byrecombinant HPV16-E7 CyaAs.

Surviving C57BL/6 mice from TC-1 grafts in the therapeutic set ofexperiments were re-grafted s.c. at day 100 with 5×10⁴ TC-1 cells.Age-matched untreated mice were taken along as controls (A). Growth oftumors in mice initially immunized with CyaA-E7₄₉₋₅₇, CyaA-E7_(Full),and CyaA-E7_(Δ30-42) are represented (B, C and D, respectively). Micewere killed when the tumor size reached 1000 mm³ or whenever thesanitary status of the animals commanded. (E) Survival curves of animals(untreated, open squares; or immunized with Cya-E7₄₉₋₅₇, open triangles;CyaA-E7_(Full), open circles; or CyaA-E7_(Δ30-42), (open diamonds) andregrafted with TC-1 cells (day of graft taken as 0). In every case, thesurvival of recombinant HPV16-E7 CyaAs-treated animals is significantlyincreased as compared to that of untreated or mock-treated mice(p<0.05).

FIG. 9. Comparison of CyaA-E7_(Δ30-42) therapeutic activity to that ofCpG ODN 1826-adjuvanted HPV16E7₄₃₋₇₇.

C57BL/6 mice were grafted s.c. on day 0 with 5×10⁴ TC-1 tumor cells.Mice were treated on days +10 and +17, with one i.d. injection of 10 μgHPV16-E7₄₃₋₇₇ (n=5, triangles), 1 μg CpG-ODN 1826 (n=5, squares), 10 μgHPV16-E7₄₃₋₇₇+1 μg CpG-ODN 1826 (n=5, diamonds), 10 μg CyaA-CysOVA (n=3,inverted triangles), or 10 μg CyaA-E7_(Δ30-42) (n=7, circles). Mice werekilled when the tumor sizes were above 1000 mm³ or whenever the sanitarystatus of the animals commanded.

FIG. 10. Analysis of the effect of pre-immunity to CyaA on the abilityof CyaA-E7_(Δ30-42) to induce TC-1 tumor rejection.

(A) C57BL/6 mice were either left untreated or immunized at day −90 orday −30, with two injections i.d. at a 7-days interval of 10 μg ofCyaAE5. At day −1, animals were bled and sera were individually assessedby ELISA for the presence of anti-CyaAE5 IgGs. Results are expressed asindividual antibody titers calculated by linear regression analysisplotting dilution versus A₄₉₂. Horizontal bars represent the medianresponse of each group. (B) Untreated (a, b), day −30 CyaAE5-immunized(c, d), and day −90 CyaAE5-immunized (e, f animals were grafted s.c. onday 0 with 5×10⁴ TC-1 tumor cells and were treated on days +10 and +17,with one i.d. injection of 10 μg CyaA-cysOVA (a, c, e), or 10 μgCyaA-E7_(Δ30-42) (b, d, f). Insets (b, d, f) are close-ups of the 0-35days period to show that all animals had palpable tumors at the timevaccination was given. Each curve represents the tumor growth in asingle animal. Mice were killed when the tumor sizes were above 1000 mm³or whenever the sanitary status of the animals commanded. In the topright of each quadrant is indicated the number of sacrificed animals vsthe total number of animals included.

FIG. 11. Induction of CTL responses in HHD mice byCyaA-HPV16E7_(Δ30-42).

The peptide loading of EL4-HHD cells is indicated above each graph. Inthe tables below are indicated the type of CyaA injected (related toHPV16E7 or HPV18E7) under the column header Vacc and the peptide usedfor in vitro restimulation under the column header Stim. As one can see,following CyaA-HPV16E7_(Δ30-42) immunization, we were able to induce CTLspecific for peptide #253 only (right panel). This CTL activity wasspecific since in vitro restimulated splenocytes with peptide #255 werenot cytotoxic towards peptide #253-coated EL4-HHD cells (right panel).The absence of CTL-specific responses towards the two others HLA-A2restricted peptide may result from different phenomenons: (i) theimmunodominance of peptide #253, (ii) the absence of processing ofpeptides #255 and #258 by the proteasome of EL4-HHD cells, (iii) thepoor solubility of peptide #258 for which we had to use acetonitrile(50%) that may be toxic for the cells. Most interestingly, co-injectionof CyaA-HPV18E7_(Δ32-42) did not interfere with the CTL inducing abilityof CyaA-HPV16E7_(Δ30-42) (right panel).

FIG. 12. Induction of CTL responses in HHD mice byCyaA-HPV18E7_(Δ32-42).

The peptide loading of EL4-HHD cells is indicated above each graph. Inthe tables below are indicated the type of CyaA injected (related toHPV16E7 or HPV18E7) under the column header Vacc and the peptide usedfor in vitro restimulation under the column header Stim. As one can see,following CyaA-HPV18E7_(Δ32-42) immunization, we were able to induce CTLspecific for peptide #251 only (left panel). This CTL activity wasspecific since in vitro restimulated splenocytes with peptide #257 werenot cytotoxic towards peptide #251-coated EL4-HHD cells (left panel). Asfor HPV16E7 HLA-A2 restricted peptides, the absence of CTL-specificresponses towards HPV18E7 #257 HLA-A2 restricted peptide may result fromdifferent phenomenons: (i) the immunodominance of peptide #251, (ii) theabsence of processing of peptide #257 by the proteasome of EL4-HHDcells, (iii) the poor solubility of peptide #257 for which we had to useacetoniitrile (50%) that may be toxic for the cells. Most interestingly,co-injection of CyaA-HPV16E7_(Δ30-42) did not interfere with the CTLinducing ability of CyaA-HPV18E7_(Δ32-42) (left panel).

FIG. 13. Construction pTRACE5-E7HPV18

FIG. 14. Construction pTRACE5-E7ΔHPV16+18

3 steps for plasmid preparation are disclosed.

FIG. 15: Map of polynucleotide encoding:

CyaA-HPV18E7_(Full)

CyaA-HPV18E7_(Δ32-42)

CyaA-HPV16+18E7_(Full)

CyaA-HPV16+18E7_(Δ)

FIG. 16: Induction of CTL by recombinant CyaAs carrying HPV18E7 inC57BL/6 mice.

FIG. 17: Induction of CTL by recombinant CyaAs carrying HPV18E7 inC57BL/6 mice.

The deposited material (I-3190 and I-3191) is contained in E. colistrain BLR and can be grown in Luria Broth (LB) medium, and seeded in LBwith 100 μg/ml Ampicillin, and can be incubated at 30° C., in air withshaking at 175 rpm and illumination. Conservation overnight is possiblewith LB in 7-10% DMSO.

EXAMPLES Example 1

Here, we constructed recombinant CyaA containing either the full-lengthsequence of the E7 protein from HPV16 or subfragments of thispolypeptide (in particular, a peptide encompassing residues 49-57 of E7that corresponds to a H-2D^(b) restricted epitope and HPV16-E7 residues43-98 plus 1-29). We showed that, when injected to C57BL/6 mice, theseHPV16-E7-recombinant CyaAs are able to induce specific CTL and Th1responses characterized by the secretion of IFN-γ. Furthermore, whentested therapeutically, these constructions were able to provide up to100% protection against the subcutaneous graft of TC-1 cells. This studyrepresents the first demonstration of the in vivo anti-tumoraltherapeutic activity mediated by CyaA against human tumor specificantigens.

Materials and Methods

Mice and Cell Lines.

Specific pathogen free 6-10-week old female C57BL/6 mice were obtainedfrom CER Janvier (Le Gesnet St-Isle, France) or Charles River(L'Arbresle, France). TAP1^(−/−) (18), MHC Class II^(−/−) (19) andCD40^(−/−) (20) bred onto a C57BL/6 background were also used in thisstudy. Animals were kept in the Pasteur Institute animal facilitiesunder pathogen-free conditions with water and food ad libitum.Experiments involving animals were conducted according to theinstitutional guidelines for animal care.

TC-1 cells expressing HPV16 E6 and E7 proteins (21) and mouse thymomaEL4 cells (17) were obtained from ATCC. Cells were maintained in RPMI1640 with Glutamax supplemented with 10% heat-inactivated FCS, 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.4 mg/ml geneticin (for TC-1 cellsonly) and 5.10⁻⁵ M 2-mercaptoethanol (Gibco BRL, Cergy-Pontoise,France).

Peptides.

The synthetic peptidesE749 57 (RAHYNIVTF (SEQ ID NO: 1), one-letter codefor amino acid) corresponding to the HPV16-E7H2-D^(b)-restricted epitope(22) and E7₄₃₋₇₇ (GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; SEQ ID NO: 2)corresponding to the E7₄₉₋₅₇ CTL epitope with its natural flankingsequence and a Th epitope (bold) (23) were purchased from Neosystem(Strasbourg, France). CpG ODN 1826 was purchased from PROLIGO (Paris,France).

Construction and Purification of Recombinant B. pertussis AdenylateCyclase Carrying HPV16-E7 Epitopes.

Recombinant adenylate cyclase used in this article were expressed in E.coli by using derivatives of plasmid pTRACE5 (FIG. 1A) which codes foran enzymatically inactive CyaA (24) (25). Plasmid pTRACE5 is anexpression vector for an enzymatically inactive, and thereforecytotoxic, variant of B. pertussis CyaA. It also expresses B. pertussisCyaC protein that is required for the postranslational acylation ofCyaA. This plasmid is a derivative of the previously described pTRACGplasmid (Gmira et al., 2001, Res. Mic. 152:889). It was obtained byinsertion of the hexanucleotide CTGCAG in the EcoRV site located withinthe 5′ part of the cyaA DNA sequence. This results in an in-frameinsertion of the dipeptide Leu-Gln between Asp188 and Ile189 of CyaAwithin an essential part of the catalytic site (Guermonprez et al. 2000,Meth. Enzymol. 326:527).

Plasmid pTRACE5 harbors a ColE1 origin of replication and an Ampicillinresistant marker. In this plasmid, the cyaC and the modified cyaA genesare placed in the same transcriptional unit under the control of the λphage Pr promoter. The pTRCAG plasmid also encodes the thermosensitive λrepressor cl⁸⁵⁷ that strongly represses gene transcription at the λ Prpromoter at temperatures below 32° C.

The E. coli strain XL1-Blue (Stratagene, La Jolla, Calif.) was used forall DNA manipulations that were performed according to standardprotocols (Maniatis et al.).

CyaA-E7₄₉₋₅₇ contains a 9-amino acid long polypeptide sequence(RAHYNIVTF; SEQ ID NO: 1) inserted between codons 224 and 235 of CyaA.The expression plasmid for CyaA-E7₄₉₋₅₇ was constructed as follows. Twosynthetic oligonucleotides (MWG, Courtaboeuf, France), BTP1 (5′-CTA GCCGTG CCC ATT ACA ATA TTG TAA CCT TTG GTA C-3′ coding strand (SEQ ID NO:5)) and BTP2 (5′-CAA AGG TTA CAA TAT TGT AAT GGG CAC GG-3′ non codingstrand (SEQ ID NO: 6)) were annealed and ligated into the pTRACE5digested with NheI and KpnI. CyaA-E7_(Full) contains the entire sequenceof the HPV16-E7 protein, i.e., 98 amino acids, inserted at the same 224position of the enzymatically inactive CyaA. The DNA sequence encodingthe E7 protein was amplified from HPV16 DNA (Seedorf K et al above)using specific primers BTP3, (5′-GGG CGC TAG CAT GCA TGG AGA TAC ACCTAC-3′; SEQ ID NO: 7), and BTP4 (5′-GGG CGG TAC CTG GTT TCT GAGAAC AGATGG G-3′; SEQ ID NO: 8). The resulting PCR product was digested by NheIand KpnI and ligated into pTRACE5 cleaved by NheI and KpnI. The SspIsite present in the annealed oligonucleotide as well as in the fullsequence of HPV16-E7 allowed rapid identification of insertion mutants.CyaA-E7_(Δ30-42) contains the first 29 amino acid residues of HPV16-E7inserted between codons 319 and 320 of CyaA as well as residues 43 to 98of HPV16-E7 inserted between codons 224 and 235 of CyaA. The expressionplasmid for CyaA-E7_(Δ30-42) was constructed in two steps. A first DNAfragment encoding (amino acid residues 1 to 29) of HPV16-E7 was PCRamplified using as a target DNA a synthetic HPV16-E7 gene (optimized forproduction in E. coli, designed by GTP Technology, Labege, France), andprimers BTP5 (5′-GGG CAC CGG TAA ACG TAT GCA CGG CGA TAC TCC G-3′; SEQID NO: 9), and BTP6 (5′-CGT GAG CAT CTG GCT TTC ACT AGT ACG TTT GTT CAGCTG CTC GTA GCA-3′; SEQ ID NO: 10). A second, DNA fragment encodingcodons 320 to 372 of CyaA was PCR amplified using pTRACE5 as target DNAand primers BTP7 (5′-GGG CAC TAG TGA AAG CCA GAT GCT CAC GCG CGG G-3′;SEQ ID NO: 11), and BTP8 (5′-AGT ACA TCC GGC GAG AAC-3′; SEQ ID NO: 12).These two DNA fragments (that partly overlap) were purified and combinedwith primers BTP5 and BTP8 in a third PCR to amplify a 294 by long DNAfragment. This fragment was digested by AgeI and BstBI and insertedbetween the corresponding sites of pTRACE5 to yield plasmidpTRACE5-E7₁₋₂₉. Then, a DNA fragment encoding the amino acid residues 43to 98 of HPV16-E7 was PCR amplified using the synthetic HPV16-E7 gene astarget DNA and primers BTP9 (5′-GGG CGC TAG CGG TCA AGC AGA ACC GGAC-3′; SEQ ID NO: 13) and BTP10 (5′-GGG CGG TAC CAG GTT TTT GAG AGC AAATCG GACAAA CAA TCC CCA GAG TAC CCA TC-3′; SEQ ID NO: 14). The purifiedPCR fragment was digested by NheI and KpnI and ligated into plasmidpTRACE5-E7129 digested by the same restriction enzymes.

All recombinant adenylate cyclase were produced in the Escherichia colistrain BLR (Novagen, Madison, Wis.) as described previously (26). Therecombinant proteins were purified close to homogeneity (FIG. 1B) frominclusion bodies by a two-step procedure that includes DEAE-Sepharoseand phenyl-Sepharose chromatography, as described previously (26). Anadditional washing step with 60% isopropanol in 20 mM Hepes-Na, pH7.5,was added to the phenyl-Sepharose chromatography in order to eliminatemost of the contaminating LPS. LPS contents were determined using thekit QCL-1000 (Biowhittaker, Walkersville, Md.). Purified recombinantproteins were analyzed by SDS-gel analysis. Protein concentrations weredetermined spectrophotometrically from the absorption at 280 nm using amolecular extinction coefficient of 142,000 M⁻¹.cm⁻¹.

Construction and Purification of Recombinant HPV16-E7 Protein.

The E. coli-optimized cDNA coding for HPV16-E7 protein (GTP technology)DNA sequence available upon request was sub-cloned in pIVEX2.4b vector(Roche Molecular Biochemicals, Meylan, France) between NcoI and XhoIrestrictions sites. The resulting plasmid was then transformed into theE. coli strain BL21λXDE3 (Novagen). The His-Tag-HPV16-E7 protein wasexpressed upon induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside(Euromedex, Souffelweyersheim, France) and purified on Ni-NTA agarose(Qiagen, Hilden, Germany) according to the manufacturer's instructions.Isopropanol washes were used as described in (27) in order to remove LPScontamination.

Immunoblotting.

Proteins were separated by SDS-PAGE and electrotransferred to anitrocellulose membrane (0.45μ, BioRad, Marnes la Coquette, France) thatwas probed with a mouse monoclonal anti HPV16 E7 antibody (Zymed, SanFrancisco, Calif.) or with a polyclonal anti E. coli BLR serum preparedin C57BL/6 mice. Immune complexes were detected with goat anti-mouseimmunoglobulins conjugated to phosphatase alkaline (Chemicon, Temecula,Calif.) and revealed with 5-bromo-4-chloro-3-indolylphosphate/nitrobluetetrazolium (BCIP/NBT) (Sigma, St. Louis, Mo.).

Mice Immunization and Tumor Rejection Experiments.

Animals were immunized with one intravenous injection of 50 μg, or withtwo intradermal injections (10 μg each) of control or HPV16-E7recombinant CyaAs diluted in PBS (Gibco BRL) Intradermal injections wereperformed in the ear dermis (47). For in vitro analysis, euthanaziedanimals (CO₂) were splenectomized 7 days after the injection except forthe analysis of long lasting responses for which this procedure wascarried out 3 months after the injection. For tumor rejectionexperiments, mice received 5×10⁴ TC-1 cells subcutaneously and weretreated by HPV16-E7 recombinant CyaAs 1 day, 5 days or 10 days aftertumor inoculation. TC-1 Tumor growth was monitored using a caliper andexpressed in cubic millimeters using the formula V=(L×w²)/2 (L: length,w: width) (48).

In Vitro Cytotoxic Assay.

Splenocytes from immunized mice were stimulated in vitro with 1 μg/ml ofeither E7₄₉₋₅₇ or E7₄₃₋₇₇ peptides in the presence of syngeneicirradiated naive spleen cells in complete medium (RPMI 1640 withGlutamax supplemented with 10% heat-inactivated FCS, 100 U/mlpenicillin, 100 μg/ml streptomycin and 5.10⁻⁵ M 2-mercaptoethanol)during 5 days. The cytotoxic activity of these effector cells was testedin a 5-h ⁵¹Cr-release assay on TC-1 cells. Radiolabeling was performedas follows: exponentially growing TC-1 cells cultured in a 7.5% CO₂atmosphere at 37° C. were quickly trypsinized (Trypsin-EDTA, GibcoBRL)and incubated with 100 μCi of ⁵¹Cr for 1 h at 37° C. Various E:T ratioswere used and all assays were performed in duplicate. The radioactivityreleased in the supernatant of each well was measured. The percentage ofspecific lysis was calculated as 100×(experimental release−spontaneousrelease)/(maximum release−spontaneous release). Maximum release wasobtained by adding 10% Triton X405 to target cells and spontaneousrelease was obtained with target cells incubated in complete mediumalone. Mice are considered as responders when at least 20% specificlysis was observed at the highest E:T ratio. Results are expressed asmedians±interquartile ranges of responder mice per group.

Single IFN-γ Producing Cell Enzyme-Linked-Immunospot Assay for SecretingCells.

Multiscreen filtration plates (96 wells; Millipore, Molshein, France)were coated with 4 μg of rat anti-mouse gamma interferon (IFN-γ)antibody (clone R4-6A2; PharMingen, San Diego, Calif.) per ml, overnightat room temperature. Then the plates were washed and blocked withcomplete medium. Serial two-fold dilutions of spleen cells fromimmunized mice were added to the wells along with 5×10⁵ γ-irradiated(2,500 rads) syngeneic feeder cells. The cells were incubated for 36 hwith or without E7₄₉₋₅₇ peptide at 1 μg/ml. After extensive washes, theplates were revealed by incubation with 5 μg of biotinylated ratanti-mouse IFN-γ antibody (clone XMG 1.2; PharMingen) per ml followed byincubation with streptavidin-alkaline phosphatase (PharMingen). Finally,spots were revealed using BCIP/NBT as the substrate. The number ofIFN-γ-producing cells was determined by counting the number ofspot-forming cells (SFC) in each well (Bioreader, Karben, Germany), andthe results were expressed as the total number of SFC per spleen (17).

Enzyme-Linked Immunosorbent Assay (ELISA).

Mice immunized intradermally with empty vector CyaAE5 were bled 30 or 90days later and individual mouse sera were tested for antibody responsesby ELISA. Microplates (Nunc, Roskilde, Denmark) were coated overnightwith empty vector CyaAE5 (3 μg/ml) in PBS. After washes in PBS-tween 20(0.1%), diluted sera were added to the wells and incubated for 1 hour at37° C. Following washes in PBS-tween 20, plates were incubated with goatanti-mouse IgG peroxidase conjugate (Sigma) for 1 hour at 37° C. Plateswere developed using o-phenylenediamine and hydrogen peroxide (Sigma).The reaction was stopped with sulfuric acid and the plates analyzed at492 nm in an ELISA reader (Dynatech, Marnes la Coquette, France).Results are expressed as antibody titers calculated by linear regressionanalysis plotting dilution versus A₄₉₂. The titers were calculated to bethe log₁₀ highest dilution that gives twice the absorbance of pooledcontrol sera diluted 1/100.

Cytokine Production.

Spleen cells from immunized mice were stimulated in vitro with 10 μg/mlof HisTag-HPV16-E7 protein or 1 μg/ml E7₄₃₋₇₇ peptide in complete mediumfor 72 hours. IFN-γ and IL-5 production was determined in culturesupernatants by sandwich enzyme-linked immunosorbent assay (ELISA) aspreviously described (28). All assays were standardized withcorresponding recombinant murine cytokines (Pharmingen).

FACS® Analysis.

TC-1 cells were processed as described elsewhere (29) for the analysisby flow cytometry of the level of expression of the MHC class I moleculeH-2 D^(b) using a specific FITC-conjugated monoclonal antibody (cloneKH95, Pharmingen, Le Pont de Claix, France).

Statistical Analysis.

Considering the small size of the different samples, non parametricstatistical tests (30) were applied using the software StatXact 4 (Cytelcorporation, Cambridge, Mass.). Survival curves were plotted using Prismsoftware (GraphPad Software Inc., Calif.) and compared with thesoftware's built-in logrank test. Data were considered significantlydifferent at p<0.05.

Results

Construction and Characterization of Recombinant Adenylate CyclasesBearing HPV16-E7 Epitopes.

To study the ability of CyaA to induce HPV16-E7 specific T cellresponses, we constructed 3 different recombinant molecules.CyaA-E7₄₉₋₅₇ contains a 9-amino acid long polypeptide sequence(RAHYNIVTF; SEQ ID NO: 1) corresponding to the previously describedH-2D^(b) restricted CTL epitope (22), that was inserted between codons224 and 235 of an enzymatically inactive (hence nontoxic) CyaA.CyaA-E7_(Full) contains the entire sequence (98 amino acids) of theHPV16-E7 protein inserted at the same 224 position of the enzymaticallyinactive CyaA CyaA-E7_(Δ30-42) contains the first 29 amino acid residuesof HPV16-E7 inserted between codons 319 and 320 of the enzymaticallyinactive CyaA as well as residues 43 to 98 of HPV16-E7 inserted betweencodons 224 and 235. To allow in vitro and in vivo assays, the CyaAconstructs were produced and purified close to homogeneity (FIG. 1B) AnLPS elimination procedure was introduced in the purification protocol(26) to obtain recombinant proteins containing less than 100 units ofendotoxin per 50 μg. The presence of the E7 protein in CyaA-E7_(Full)and CyaA-E7_(Δ30-42) was confirmed by western blotting using a specificmonoclonal antibody (Zymed) (FIG. 1C). In contrast, CyaA-E7₄₉₋₅₇containing only the H-2D^(b) restricted epitope was not recognized bythe anti-HPV16-E7 antibody. The overall biochemical properties of the 3recombinant CyaAs were not modified and these molecules displayed ahemolytic activity similar to that of wild type adenylate cyclase (17).

Immunization with HPV16-E7 Recombinant CyaAs Induces E7-Specific CTLResponses.

To test whether CyaA can induce CTL responses against HPV16-E7 epitopes,C57BL/6 mice were immunized once, intravenously with 50 μg of thedifferent HPV16-E7 recombinant CyaAs. Splenocytes were harvested andstimulated in vitro with 1 μg/ml of the E7₄₃₋₇₇ peptide. Their abilityto lyse TC-1 cells was determined 5 days later using a ⁵¹Cr releaseassay. As shown in FIG. 2A, a single i.v. immunization of C57BL/6 micewith HPV16-E7 recombinant CyaAs induced strong and specific CTLresponses to TC-1 cells. Immunization with CyaA containing the fullHPV16-E7 protein (FIG. 2A, b) or its deleted form, CyaA-E7_(Δ30-42)(FIG. 2A, c), resulted in higher maximal CTL activities as compared tothat induced by CyaA-E7₄₉₋₅₇ that contains only the minimal H-2D^(b)restricted epitope (FIG. 2A, a), although we could not demonstratestatistical significance from our data. Similar results were obtainedwhen peptide E749-57 was used for in vitro restimulation (data notshown). Splenocytes from mice vaccinated with a recombinant CyaAcarrying a non relevant epitope(OVA₂₅₇₋₂₆₄) and restimulated in vitrowith 1 μg/ml E7₄₃₋₇₇ peptide yielded only a weak non specific TC-1 celllysis (FIG. 2A, a). It has been previously shown that the delivery ofthe OVACD8⁺ cell epitope (SIINFEKL; SEQ ID NO: 15) to the MHC class Imolecule by CyaA in vivo was dependent on TAP1 function (15). We testedwhether this is also the case using CyaA-E7_(Δ30-42). As shown in FIG.2A, d, in vitro stimulated splenocytes from i.v. vaccinated TAP1^(−/−)mice, were unable to lyse TC-1 cells. We also tested the requirement ofCD4⁺ T cell help in the in vivo priming of CTL response byHPV16E7-containing recombinant CyaA. In agreement with earlierobservations (15), we observed that i. v. vaccination of MHC Class11^(−/−) mice using CyaA-E7_(Δ30-42), resulted in the induction of highlevel of specific CTL responses to TC-1 cells (FIG. 2A, e). In contrast,we observed in this model some dependence towards CD40 signaling, as weobserved a low level of CTL response to TC-1 cells in CD40^(−/−) mice(FIG. 2A,f).

To estimate ex vivo the frequencies of HPV16-E7-specific splenocytes inmice immunized with recombinant CyaAs, the number of cells producingIFN-γ in response to in vitro stimulation with HPV16-E7₄₉₋₅₇ peptide wasquantified by enzyme-linked immunospots (ELISPOT). FIG. 2B shows thatthere was only a slight difference in the number of IFN-γ-producingsplenocytes specifically producing IFN-γ obtained from mice immunizedwith CyaE5-CysOVA as compared to those immunized with CyaA-E7₄₉₋₅₇. Incontrast, the number of IFN-γ-producing splenocytes obtained was muchmore higher (p<0.05) in mice vaccinated with HPV16-E7 recombinant CyaAscontaining either the full HPV16-E7 protein or its deleted form. Theobserved responses were epitope specific as very few spleen cells fromthese mice produced IFN-γ in the absence of stimulation by theHPV16-E7₄₉₋₅₇ peptide (FIG. 2B). These results show that CyaA is able todeliver in vivo the immunodominant CD8⁺ H-2D^(b)-restricted T-cellepitope of the HPV16-E7 protein into the cytosol of immunocompetentcells for processing and presentation into the MHC class I pathway, toelicit strong CTL responses. In accordance with previous observations(25, 31), we confirm that CyaA is tolerant to insertion of largepolypeptidic fragments as CyaAs carrying the full HPV16-E7 protein orits deleted and inverted form, were also able to induce strong CTLresponses. Our data also demonstrate that these latter molecules inducedsignificantly higher frequencies of HPV16-E7₄₉₋₅₇-specific responses inmice.

Immunization with HPV16-E7 Recombinant CyaAs Induces HPV16-E7 SpecificTh1 Responses.

Th1 responses play an important role in protection against intracellularpathogens and tumor development (32, 33). We therefore characterized thetype of T-cell responses induced by HPV16-E7 recombinant CyaAs. C57BL/6mice were immunized once i.v. with 50 μg of the three different HPV16-E7recombinant CyaAs, and cytokine synthesis was determined after in vitrostimulation of spleen cells with 10 μg/ml of the purified His-TagHPV16-E7 protein. As shown in FIG. 3, immunization with CyaAs carryingthe full HPV16-E7 protein or its deleted form resulted in a Th1-likeprofile characterized by the production of high levels of IFN-γ and thelack of detectable levels of IL-5. This response was specific sinceIFN-γ levels obtained after immunization with CyaA-E7_(Full) orCyaA-E7_(Δ30-42) were significantly higher than those obtained in micemock-immunized with CyaAE5-CysOVA (p<0.05). However, this was not thecase with splenocytes from mice immunized with CyaA-E7₄₉₋₅₇. Similarresults were achieved when the restimulation was carried out with 1μg/ml of E7₄₃₋₇₇ peptide (FIG. 3A, inset).

Taken together, these results indicate that, in our conditions, CD4⁺ Tcells play an important role in the secretion of IFN-γ as levelsobtained with CyaAs carrying the full HPV16-E7 protein which containsclass II H-2^(b) restricted T cell epitopes, are much higher than thoseobtained with CyaA-E7₄₉₋₅₇ which contains only the class I H-2D^(b)restricted epitope.

Immunization with HPV16-E7 Recombinant CyaAs Induces Regression ofEstablished HPV16-Expressing Tumors.

Considering the robust immunological responses obtained, we thenevaluated in vivo the therapeutic activity of HPV16-E7 CyaAs in apre-clinical model consisting of a H-2^(b) tumorigenic cell lineexpressing HPV16-E6 and E7 proteins (TC-1 cells) which is injectedsubcutaneously to C57BL/6 mice. In this model, it has been previouslyshown that tumor rejection is exclusively mediated by E7₄₉₋₅₇-specificCD8⁺ T-cells (21, 22, 34, 35). Thus, 5×10⁴ TC-1 cells were injected s.c.in the right flank of C57BL/6 mice and 50 μg of CyaAE5-HPV16-E7₄₉₋₅₇,-E7_(full) or -E7_(Δ30-42) were injected intravenously to mice 1, 5 or10 days later. FIG. 4 represents the tumor growth in mice treatedtherapeutically 10 days after tumor grafting. Of note, in theseconditions 100% of the animals developed palpable tumors by the timevaccination was given. To avoid unnecessary suffering, animals weresacrificed when tumor sizes reached 1000 mm³. All untreated animals, aswell as mice who were treated with a mock CyaAE5-cysOVA developed tumorsof that size (>1000 mm³) within a maximum of 49 days. In sharp contrast,the majority of animals treated with HPV16-E7 recombinant CyaAs remainedtumor free throughout the duration of the experiment (FIG. 4, C, D, E.FIG. 5 shows the plot of the survival of the animals grafted with TC-1cells and submitted to three different therapeutic protocols in whichthe recombinant CyaAs were injected at either day 1, day 5 or day 10after the TC-1 graft. The medians survival times of untreated ormock-treated animals were comprised between 31 to 40 days. In contrast,the survival of mice vaccinated with CyaAs carrying HPV16-E7 antigenswas significantly superior to that of control animals (p<0.05).Differences in the protective activities of the various constructs couldbe established although the statistical significance could not, becauseof the small size of the different samples. If the rate of tumorregression conferred by CyaA-E7₄₉₋₅₇ and -E7_(Full) could not benoticeably differentiated, CyaA-E7_(Δ30-42) was clearly superior interms of tumor regression and growth inhibition, since in all the threetherapeutic schemes, the protection rate was always higher than 90%.

Some animals vaccinated with CyaA-E7₄₉₋₅₇ and CyaA-E7_(Full) appeared togrow tumors lately in the time course of the experiment (FIG. 4 (*) anddata not shown). Bearing in mind that this phenomenon could reflecttumor escape mechanisms, we explanted the growing tumors from theseanimals and analyzed these cell lines named TC-1 A1 and TC-1 A2 by FACSanalysis for H-2D^(b) expression. As shown in FIG. 6, as compared totheir native counterpart, TC-1 A1 and A2 cells explanted from lategrowing tumors had lost the expression of H-2D^(b), thus most probablyrendering them undetectable to E7₄₉₋₅₇-specific CD8⁺ T-cells. Since TC-1cells grew in vivo in an environment without antibiotic pressure forselection, we also checked by western blot for the expression ofHPV16-E7 in TC-1 A1 and A2 cells. We could not find any difference inexpression of this protein between TC-1 and TC-1 A1 and A2 cells (datanot shown).

Taken together, these results demonstrate the efficiency of theadenylate cyclase vector as a suitable therapeutic vaccine for inducingthe regression of HPV16-expressing tumors in a pre-clinical model.

We tested another injection route of clinical interest. Hence, 10 μg ofCyaA-E7_(Δ30-42) were injected i.d. twice at a 7-days interval starting10 days after TC-1 graft. Interestingly, as all untreated andmock-treated animals developed tumors, we observed tumor regression inall of the animals treated with CyaA-E7_(Δ30-42) (FIG. 4B, a, b). Thistherapeutic immunization resulted in a 100% survival at 90 days of theCyaA-E7_(Δ30-42)-treated mice whereas the survival medians of untreatedand mock-treated animals were 30 and 32 days, respectively (FIG. 4, c).

Long Term Persistence of HPV16-E7₄₉₋₅₇ Specific CD8⁺ T-Cells Induced byCyaA Immunization.

To assess the persistence of immune response induced by HPV16-E7recombinant CyaAs, mice surviving from therapeutic experiments after 3months were sacrificed and their splenocytes subjected to in vitrostimulation for five days with 1 μg/ml E7₄₃₋₇₇ peptide. Their ability tolyse TC-1 cells was then determined by a ⁵¹Cr release assay. As shown inFIG. 7, specific CTL responses to HPV16-E7₄₃₋₇₇ peptide were stilldemonstrated from splenocytes of animals immunized three months ago.Based on the maximum percentage of specific lysis at 30:1 ratioeffector:target, the immunological response appeared to be more robustin animals treated with CyaA-E7₃₀₋₄₂, although no statisticalsignificance could be demonstrated from these data. To assess thephysiological relevance of such a long lasting immunogenicity, remaininganimals were re-challenged s.c. with 5×10⁴ TC-1 cells at day 100. Undersuch conditions, all naive age-matched control animals developed tumorsand displayed a survival median time of 37.5 days (FIG. 8). In contrast,mice immunized three months ago with HPV16-E7 recombinant CyaAs werevery significantly protected from tumor development. As observed above,animals vaccinated with CyaAE5-HPV16-E7_(Δ30-42) displayed a high levelof protection. However, at variance with results obtained in the firstset of therapeutic experiments, there is now a striking difference ofprotection between animals treated with CyaA-E7₄₉₋₅₇ and those treatedwith CyaA-E7_(Full) in favor of the latter, although the small size ofthe samples did not allow to unequivocally demonstrate a statisticalsignificance. These observations suggest that in this model, T-cell helpprovided by CyaA carrying the full HPV16-E7 protein is of importance forefficient long lasting responses against TC-1 cells.

Taken together, our results demonstrate that one i.v. injection of 50 μgof recombinant CyaAs bearing the helper epitope of the HPV16-E7 protein(DRAHYNIVTF; SEQ ID NO: 16) is sufficient to induce long lasting E7₄₉-57specific CD8⁺ T cells that are capable of conferring protection againsttwo TC-1 tumor cells challenges over a period of time of at least 6months.

Therapeutic Efficacy of CyaA-E7_(Δ30-42) Compares Favorably to that ofPeptide Administered with CpG-ODN 1826.

To better evaluate the potency of CyaA as an antigen delivery system, wecompared the therapeutic efficacy of CyaA-E7_(Δ30-42) to that ofHPV16-E7₄₃₋₇₇ peptide supplemented with CpG-ODN 1826 (37). Mice weretherefore injected s.c. with 5×10⁴ TC-1 cells and treatedtherapeutically 10 and 17 days later via the intra-dermal route with 10μg of CyaA-E7_(Δ30-42) or 10 μg of HPV16-E7₄₃₋₇₇ peptide administeredwith 1 μg of CpG-ODN 1826. The survival rates were similar in these twogroups (FIG. 9), although results obtained with CyaA-E7_(Δ30-42) wereslightly better but not statistically different from those obtained withHPV16-E7₄₃₋₇₇ peptide mixed with CpG-ODN 1826. Of note, this result wasobtained using 50 times more HPV16-E7₄₃₋₇₇ peptide than CyaA-E7_(Δ30-42)on a molar basis. When used alone, the peptide HPV16-E7₄₃₋₇₇ had noeffect on TC-1 tumor growth.

Prior Immunity to CyaA Vector Marginally Affects the TherapeuticEfficacy of CyaA-E7_(Δ30-42).

In a clinical setting, multiple boosts will probably have to be given topatients with lesions in order to obtain efficient cellular immuneresponses. It is therefore essential to demonstrate that pre-immunity tothe CyaA vector does not impair its ability to trigger tumor rejection.To do so, we immunized mice i.d. twice at a 7-days interval with 10 μgof empty vector CyaAE5, 90 or 30 days prior to s.c. injection with 5×10⁴TC-1 cells. Therapeutic treatment with two i.d. injections at a 7-daysinterval of 10 μg CyaA-E7_(Δ30-42) was set on day 10. Analysis ofantibody responses showed that empty vector immunized-mice were immuneto CyaA at the time of TC-1 injection (FIG. 10A). We then compared theability of CyaA-E7_(Δ30-42) treatment to induce tumor rejection inage-matched naive animals and in CyaA immune animals. Whatever theirimmune status towards CyaA, the majority of mice treated withCyaA-E7_(Δ30-42) remained tumor free throughout the experiment (FIG.10B). Only 1 animal in the day-30 immune mice group and 2 in the day-90immune mice group developed tumors (FIG. 10B b, d, f). In contrast, 100%of mock-treated animals developed tumors and were sacrificed (FIG. 10B,a, c, e). We did not observe any correlation between the level ofanti-CyaA antibodies titers and the development of TC-1 tumors (data notshown). Furthermore, survival curves of the CyaA-E7_(Δ30-42)-treatedmice (FIG. 10B, b, d, f) were not statistically different (p=0.324).

These data therefore indicate that the effect of pre-existing immunitytowards CyaA has only very limited effect on the ability of this vectorto subsequently induce efficient responses against a foreign givenantigen.

DISCUSSION

Previous studies have demonstrated that the adenylate cyclase fromBordetella pertussis is a powerful tool to deliver in vivo, CD4⁺ andCD8⁺ T cell epitopes to the MHC-class II and I presentation pathways ofdendritic cells. In experimental murine models, this system has beenused to trigger efficient Th1 and CTL responses providing anti-viral andanti-tumoral protection (36). As an evaluation of the potentialapplication of CyaA in humans for the treatment of HPV16-associatedcervical malignancies, we went on to demonstrate that this vectorefficiently delivers in vivo epitopes from the E7 protein from HPV16.

We constructed various HPV16 recombinant CyaAs containing the full E7protein from HPV16 or sub-fragments of this polypeptide, including inparticular the H-2D^(b)-restricted minimal CTL epitope corresponding toresidues 49-57. We showed that these different recombinant proteins wereable to prime specific and strong CTL responses when injected to C57BL/6mice. Our data confirmed that the delivery of CTL epitopes by CyaArequires a fully functional Class I presentation pathway as we could notprime CTL upon injection of CyaA-E7_(Δ30-42) in TAP1^(−/−) mice (15).The CTL priming mediated by CyaA was independent of the presence of CD4⁺T cells as indicated by the efficient CTL responses obtained in MHCClass II^(−/−) mice in agreement with previous results (15). Thischaracteristic of CyaA as a vaccinal vector is of great importance whenconsidering the vaccination of immunosuppressed or immunodeficientpatients presenting an altered number of CD4⁺ T cells. However, low CTLresponses were obtained in CD40^(−/−) mice indicating that CTL primingwas partially dependent upon CD40 signaling. These observations suggestthat HPV16-E7 recombinant CyaAs elicit MHC class I-restricted CTLdirectly by direct stimulation of professional APC. CD40-CD40Linteraction is nonetheless required to obtain optimal priming of CTLresponses.

We compared the immunogenicity of the minimal H-2D^(b)-restricted CTLepitope of HPV16-E7 to that of the full or the Δ30-42 E7 protein whichcontain among probably others, the described helper epitope DRAHYNIVTF(SEQ ID NO: 16) (37). The CTL priming as well as the frequencies ofHPV16-E7-specific splenocytes induced by CyaA-E7_(Full) andCyaAE-E7_(Δ30-42) were superior to those induced by CyaA-E7₄₉₋₅₇carrying only the CTL epitopes E₄₉₋₅₇. These observations indicate thatsimultaneous delivery of CTL and Th epitopes by CyaA results in morerobust CTL response. This is in agreement with previously published datain other models at the pre-clinical (37) and clinical level (38). Thedelivery of HPV16-E7 Th epitopes by recombinant CyaAs was furtherevidenced by the analysis of the cytokine produced by HPV16-E7-specificsplenocytes restimulated in vitro with recombinant HisTag-HPV16-E7protein or E7₄₃₋₇₇ peptide. Indeed, we observed specific synthesis ofIFN-γ only in mice vaccinated with recombinant HPV16-E7 CyaAs containingthe Th epitope. The typical Th1 profile characterized by a high level ofIFN-γ and no secretion of IL-5, that we observed after one i.v.immunization with CyaA-E7_(Full) and CyaA-E7_(Δ30-42) highlights thepotential interest of this vector for tumor immunotherapy.

This was tested in a tumor rejection model based on s.c.-establishedtumorigenic TC-1 cells (21). In accordance with our data demonstratingthe immunogenicity of HPV16-E7 recombinant CyaAs, we observed that theserecombinant proteins were able to induce the regression of establishedTC-1 tumors. CyaA-E7₃₀₋₄₂ was superior to CyaA-E7₄₉₋₅₇ andCyaAE5-HPV16-E7_(Full) in terms of survival over a period of 90 days. AsCyaAs carrying the full and the Δ30-42 E7 protein yielded comparableresults in terms of CTL priming capacity, frequencies of HPV16-E7₄₉₋₅₇specific splenocytes and production of IFN-γ, we expected CyaA-E7_(Full)to be superior to CyaA-E7₄₉-57 in terms of survival. A higher number oftested animals would have, most probably, help to rule out this apparentdiscrepancy. Yet, two aspects regarding the biochemistry of CyaA shouldbe discussed here. First, the presence of negatively charged amino acidin the region 224-235 of CyaA was shown to inhibit the translocation ofthe catalytic domain of CyaA in the cytosol of eukaryotic cells (39). Inthis respect, the acidic E7 protein (pKi=4.17) might have precluded anefficient translocation of the N-terminal domain of CyaA into thecytosol of DC. CyaA-E7_(Δ30-42) has been specifically designed to removea stretch of negatively charged amino acids located between residues 30to 42 (DSSEEEDEIDGPA; SEQ ID NO: 17) in order to favor its delivery intoDC (and furthermore two positively charged amino acids (KR) wereintroduced at each side of the inserted N-terminal domain of HPV16-E7.Second, Gmira et al. (25) have demonstrated that unfolding of theheterologous protein inserted within CyaA is mandatory to allowinternalization of the recombinant protein into the target cells. Theinsertion of two different fragments of the E7 polypeptide into twodifferent permissive sites in CyaA-E7_(Δ30-42) may prevent E7 foldingand thus should facilitate its translocation into target cells.

In the time course of tumor rejection experiments, some mice startedlately to grow HPV16 positive tumors after having previously rejectedestablished TC-1 tumors. FACS® analysis revealed that cells from thesetumors did not express H-2D^(b) molecules. This observation leads toconsider the possibility of a homologous boosting of recombinant CyaAvaccination so as to eradicate more strikingly tumor cells and toprevent tumor relapse via escape mechanisms. Bearing in mind data fromother teams in the field (37, 40, 41), it would be relevant to boostCyaA vaccination in mice rising the total amount of injected recombinantHPV16-E7 CyaA to 100 μg, i.e. 0.56 nmoles. Experiments intended to testthis latter observation, as well as others to test different ways ofCyaAs administration are being conducted.

Upon re-challenge with TC-1 cells, surviving mice immunized withHPV16-E7 recombinant CyaAs were selectively protected. This wascorrelated with the presence of HPV16-E7₄₉₋₅₇ CD8⁺ T-cells among thesplenocytes of these animals. This observation strengthened the factthat late relapses observed in FIG. 4, were due to tumor escapemechanisms and not to waning immunity towards the E7 protein. The bettersurvival rate of mice immunized with recombinant CyaAs containing Thepitopes indicated that providing T-cell help against the same antigenalso results in an efficient recall of HPV16-E7₄₉₋₅₇ CD8⁺ T-cells. Inthis respect, it has been proposed that CD4⁺ T cells, through CD40L mayimprint a unique molecular signature on effector CD8⁺ T cells, endowingthem with their capacity for improved cell function (42).

In a validated model for the testing of novel immunotherapeutics for thetreatment of HPV-associated neoplasia (21), we have demonstrated thatCyaA is an efficient vector to induce the regression of establishedtumors as well as to provide protection against tumorigenic challengefor over a long period of time. Unlike other approaches that arecurrently developed (11), CyaA-based immunotherapy precludes the need toselect HLA restricted epitopes as full proteins can be inserted, andavoids the use of viral vectors and/or potentially oncogenic HPV DNAsequences. Furthermore, we obtained best results with a CyaA thatcontains two sub-fragments of HPV-E7 inserted into two differentpermissive sites of CyaA.

The fact that this latter construction includes all the HPV16-E7 HLAclass I and class II epitopes described in the literature (8, 46)strengthens the selection of CyaA-E7_(Δ30-42) in vaccine applications.

Based on the data presented here, we plan to test the efficacy of CyaAcontaining HPV16-E7 in clinical trials targeting cervical and analdysplasias associated with HPV infection.

Example 2

The objective was to make a bivalent therapeutic vaccine that targetboth HPV16 and HPV18 E7 proteins to treat HPV16 and HPV18-associatedmalignancies in humans. Vaccine candidates termedCyaAE5-HPV16E7_(Δ30-42) and CyaAE5-HPV18E7_(Δ32-42) have therefore beendesigned, constructed, produced and purified. HHD mice are H-2D^(−/−),β2m^(−/−) double-knockout mice expressing the HHD transgene comprisingthe α1 (H) and α2 (H) domains of HLA*0201 linked to the α3 transmembraneand cytoplasmic domains of H-2D^(b) (D), with the α1 domain linked tohuman β2-microglobulin. Thus, the only MHC class I molecule expressed bythe HHD mice is the modified HLA*0201 molecule (Pascolo et Lemonnier).

The goal of this experiment was to demonstrate:

-   -   1—that recombinant CyaA is able to deliver HLA-A2 restricted        epitopes of HPV16 and HPV18 E7 proteins.    -   2—There is no phenomenon of immuno-dominance of one HPV        recombinant cyaA over the other.

To do so, HHD mice were vaccinated i.v. with 50 μg ofCyaAE5-HPV16E7_(Δ30-42) (3 mice), CyaAE5-HPV18E7_(Δ32-42) (3 mice) orwith CyaAE5-HPV16E7_(Δ30-42)+CyaAE5-HPV18E7_(Δ32-42) in the same(retro-orbital) injection (50 μg in 200 μl) (5 mice). Seven days later,pooled splenocytes were restimulated in vitro with HLA-A2 peptides fromHPV16E7 or HPV18E7 either described in the literature or estimated fromthe SYFPEITHI software.

CTL activity was assayed 5 days later using ⁵¹Cr release. Targets cellswere HHD-EL4 cells, loaded or not with the different relevant peptides.

Results show that, vaccination with CyaAE5-HPV16E7_(Δ30-42) orCyaAE5-HPV18E7_(Δ32-42), induced the specific lysis of EL4-HHD cellsloaded with HPV16E7 or HPV18E7 peptide following autologous peptidic invitro restimulation.

This result also demonstrates that co-injection ofCyaAE5-HPV16E7_(Δ30-42) together with CyaAE5-HPV18E7_(Δ32-42) does notimpair the immunogenicity of any of the HPV recombinant CyaA as similarresponses to relevant peptides were observed.

This result demonstrates that CyaAE5-HPV16E7_(Δ30-42) andCyaAE5-HPV18E7_(Δ32-42) are able to induce in vivo a cytotoxic responseto human HLA-A2 restricted epitopes of respective E7 protein.

Mice

Specific pathogen free HHD mice were bred at the Pasteur Institute.Eleven 6-10-week old males were used for this experiment.

Reagents and Biological Material

Reagents and Buffers

-   RPMI 1640 medium-glutamax (invitrogen GIBCO, ref: 6187010)-   Ethanol 70° (Prolabo, ref: MC311631)-   Penicillin-streptomycin (invitrogen GIBCO, ref: 15140122)-   Foetal Bovine Serum (FBS) (PERBIO, ref: CH30160.03)-   β mercapto-ethanol (BIO-RAD, ref: 161-0710)-   Pyrolyzed water-   Blue trypan (SIGMA, T-8154)-   51Cr-   Trilux Scintillant (Wallac)

Peptides

Five synthetic peptides (Neosystem, Strasbourg, France) were used in forin vitro stimulation of splenocytes prior to the ⁵¹Cr release assay:

-   -   E7₁₁₋₂₀(YMLDLQPETT (SEQ ID NO: 18), one-letter code for amino        acid, #253) corresponding to a HPV16-E7 HLA-A2-restricted        epitope (1),    -   E7₈₂₋₉₀ (LLMGTLGIV (SEQ ID NO: 19), #258) corresponding to        aHPV16-E7 HLA-A2-restricted epitope (1),    -   E7₈₆₋₉₃ (TLGIVCPI (SEQ ID NO: 20), #255) corresponding to a        HPV16-E7 HLA-A2-restricted epitope (1),    -   E7₇₋₁₅ (TLQDIVLHL (SEQ ID NO: 21), #251) corresponding to a        HPV18-E7 HLA-A2-restricted epitope predicted by SYFPEITHI        software,    -   E7₈₆₋₉₄ (FQQLFLNTL (SEQ ID NO: 22), #257) corresponding to a        HPV18-E7 HLA-A2-restricted epitope (2).

Peptides have been diluted at 1 mg/ml in sterile, apyrogenic water(#253, 255, 251) or apyrogenic water, 0.1 M NaHCO₃; acetonitrile (50/50)(#257 and 258).

Recombinant Adenyl Cyclases (CyaA)

Two CyaA have been tested in this experiment:

-   -   CyaA-HPV16E7_(Δ30-42)    -   CyaA-HPV18E7_(Δ32-42)

These CyaA were expressed in E. coli strain BLR by using derivatives ofplasmid pTRACE5 coding for an enzymatically inactive CyaA (CyaAE5).Construction of plasmids, production and purification of all recombinantproteins are made as described above in Example 1 relating toconstruction and purification of recombinant B. pertussis adenylatecyclase carrying HPV16-E7 epitopes, however with the epitopes used forthe present experiment.

Stock solutions (stored at −20° C.) containing respectively 1.22 mg/mland 1.33 mg/ml of CyaA in 8 M urea, were thawed and diluted at 250 μg/mlwith PBS (Gibco BRL) before i.v. administration to mice. The finalconcentration of urea was thus between 1.6 and 1.5 M, respectively.

Cell Lines

EL4-HHD cells were used as targets in ⁵¹Cr CTL assay.

These cells were maintained in complete medium RPMI 1640 with Glutamaxsupplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100μg/ml streptomycin, and 5.10⁻⁵ M β2-mercaptoethanol (Gibco BRL,Cergy-Pontoise, France).

Methods

Mouse Immunization

Vigil animals were immunized with one intravenous (retro-orbital)injection (50 μg in 200 μl) of CyaAE5-HPV16E7_(Δ30-42) and/orCyaAE5-HPV18E7_(Δ32-42) with 0.3 ml insulin Syringes (Terumo).

Experimental Plans

The table 1 describes vaccine candidates and treatments administered toeach group of mice.

TABLE 1 Experimental group definition: CTL induction Induction of CTL(CTL Assay) Number Immunization dose/ of mice CyaAE5 Volume/Ureaconcentration 3 CyaA-HPV16E7_(Δ30-42) 50 μg/200 μl/1.6 M 3CyaA-HPV18E7_(Δ32-42) 50 μg/200 μl/1.5 M 5 CyaA-HPV16E7_(Δ30-42) 50 μgof each/200 μl/3.1 M and CyaA- HPV18E7_(Δ32-42)

CTL Assay

For in vitro evaluation of the cellular immune response induced byimmunization, euthanized animals (CO₂) were splenectomized 7 days afterimmunization. Splenocytes from each group were pooled before CTL assay.

Stimulation of CTLs (Prepare 2 Flasks T25 per Spleen)

-   Sample spleens and crush them in RPMI 1640-Glutamax 1% Ab.-   Decant-   Centrifuge at 1200 rpm for 10 minutes.-   Resuspend cells in 2 ml RMPI 1640-Glutamax 1% Ab-10% FCS-5.10-5M (β    mercapto-ethanol (complete medium: CM)-   In each T25 Flask, put:-   10 ml of CM-   5.10⁷ effector cells-   Relevant peptide at 10 μg/ml final concentration

Incubate for 5 days without moving the flasks

Cytotoxicity Assay

1/Target Cells:

-   The day before, dilute target cells 1/3 or 1/2 so as to harvest them    in exponential growth-   Transfer to 15 ml tubes.-   Centrifuge at 1200 rpm for 10 minutes-   Resuspend in 1 ml of RPMI 1640-1% Ab-   Numerate.-   Prepare 2 tubes: one with peptide, one without peptide.-   Resuspend in 150 μl:    -   peptide (50 μmolar) in one of the two tubes.    -   100 μCi of ⁵¹Cr (50 μl) for 3 10⁶ cells.    -   Medium up to 150 μl.-   Incubate for 1 hour in waterbath at 37° C., gently shake every 15    minutes.    2/Effector Cells-   Discard about 5 ml of supernatant with pasteur pipette.-   Resuspend thoroughly cells by pipetting.-   Centrifuge at 1200 rprm for 10 minutes-   Resuspend in 1 ml of CM. Numerate.-   Adjust to 10⁷ cells/ml.-   Dilute so as to obtain following effector/target ratios: 200/1,    100/1, 50/1, 25/1, 12/1, 6/1-   Distribute 100 μl/well in U-bottom microtiter plate.-   Incubate at 37° C. 7.5% CO₂ while finishing the preparation of    target cells    3/Assay:-   Wash target cells with 10 ml of RPMI 1640-1% Ab-   Wash again with 10 ml of CM.-   Resuspend in 2 ml of CM-   Numerate-   Adjust to 10⁵ cells/ml-   Distribute 100 μl/well.-   Prepare 6 wells for spontaneous release (Adjust to 100 μl in CM)-   Prepare 6 wells for maximum release (Add 100 μl of 20% Triton X-405)-   Incubate at 37° C. 7.5% CO₂ for 4 to 5 hours.    4/Counting:-   Centrifuge 5 to 10 minutes at 2000 rpm.-   Put 100 μl of Trilux scintillant in a floppy P96 microtiter plate-   Sample 50 μl of supernatant and transfer it to the floppy plate.-   Seal the plate with plastic film tape.-   Count the following day in a wallac counter.

HPV16E7 CTL Specific Responses

CTL responses induced by CyaA-HPV16E7_(Δ30-42) is shown in FIG. 11.

HPV18E7 CTL Specific Responses

CTL responses induced by CyaA-HPV18E7_(Δ32-42) is shown in FIG. 12.

Conclusion

Both CyaA-HPV16E7_(Δ30-42) and CyaA-HPV18E7_(Δ32-42) are able to inducespecific CTLs towards respective HLA-A2 restricted peptides in thischimeric HHD model.

Co-injection of CyaA-HPV16E7_(Δ30-42) and CyaA-HPV18E7_(Δ32-42) did notinterfere with each individual CyaA's ability to induce CTLs againstrespective specific HLA-A2 restricted peptides. This indicates thatthere is no phenomenon of immunodominance of one construction towardsthe other. This constitutes a crucial observation in regards to thestrategy to make-up a bivalent vaccine.

Example 3 Construction and Immunological Evaluation of RecombinantAdenylate Cyclases Containing the E7 Protein from HPV18 and the E7Proteins from HPV16 and HPV18

Carcinomas of the anogenital tract account for nearly 12% of all cancersin women, making cervical carcinoma (CxCa) the second most frequentgynecological cancer in the world. The critical observation thatinfection with human papillomavirus (HPV) might be the causative agentfor CxCa was subsequently confirmed by epidemiological studies. The mostprevalent HPV types associated with CxCa are HPV16 and HPV18 (55% and12% prevalence, respectively) {Clifford, 2003}. In order to cover alarger population, it was decided to construct a bivalent therapeuticvaccine carrying E7 from HPV16 and HPV18. Two possible strategies havebeen tested; (i) the first one to mix equimolar quantities of tworecombinant CyaAs carrying E7 from HPV16 on one hand and E7 from HPV18on the other; (ii) the second one is to construct a recombinant CyaAcarrying both E7 proteins from HPV16 and HPV18. The present reportdescribes the construction of recombinant CyaAs carrying E7 from HPV18or E7 from both viruses. The immunogenicity of the constructions weretested in a CTL assay.

Epitope Identification

The amino-acid sequence of HPV18E7 was submitted to SYFPEITHI whilesearching for H-2^(b) epitopes. This software uses an algorithm topredict epitopes that may bind to a defined MHC molecule. The softwarereturned a putative epitope restricted by the H-2D^(b) molecule with ascore of 25. Usually we consider epitopes when their score is above 22.The putative peptide IDGVNHQHL (SEQ ID NO: 3) was therefore synthesizedby Neosystem.

Constructions of Recombinant CyaAs

The scheme representing the strategy use to construct the plasmids toproduce CyaA-HPV18E7, CyaA-HPV18E7_(Δ32-42), CyaA-HPV16+18E7,CyaA-HPV16+18ΔE7, are shown in FIGS. 13 and 14.

CTL Assay

The goal of this experiment was to demonstrate the in vivo induction ofCTL by recombinant CyaA carrying the either fragments or the entireHPV18E7 protein as well as both HPV16 and HPV18 E7 proteins. TheH-2D^(b) epitope targeted are HPV18E7₄₁₋₄₉ (IDGVNHQHL; SEQ ID NO: 3)obtained from computer predictive software and HPV16E7₄₉₋₅₇. C57BL/6mice were vaccinated i.v. with 50 μg of CyaA-CysOVA, CyaA-HPV18E7,CyaA-HPV18E7_(Δ32-42), CyaA-HPV16+18E7 or CyaA-HPV16+18ΔE7 (2 in eachgroup). 7 days later, pooled splenocytes were restimulated in vitro withpeptides HPV18E7₄₁₋₄₉ (10 g/ml), OVA257-264 (1 g/ml) or HPV16E7₄₃₋₇₇ (1μg/ml). CTL activity was assayed 5 days later using 51 Cr release.Targets cells were EL4 cells loaded or not with HPV18E7₄₁-49 peptide(8-nmol) or TC-1 cells. The legend in the figures indicate:vaccination/restimulation. Example: OVA/OVA refers to mice vaccinatedwith CyaAE5-CysOVA and restimulated with the OVA peptide.

These results (FIG. 16) show that, in contrast to CyaA-HPV18E7_(Full),CyaA-HPV18E7Δ₃₂₋₄₂ is able to deliver in vivo a computer predictedCD8+H-2D^(b)-restricted T-cell epitope of the HPV18-E7 protein into thecytosol of immunocompetent cells for processing and presentation intothe MHCclass I pathway, to elicit strong CTL responses. It is odd thatCyaA-HPV18E7Δ₃₂₋₄₂ is able to prime CTL response against HPV18E7₄₁₋₄₉because this recombinant CyaA lacks the first two amino acids of thepeptide HPV18E7₄₁₋₄₉. However at the insertion site of HPV18E7 fragment43 to 105, there are one alanine and one serine so that the putativepeptide is now ASGVNHQHL (SEQ ID NO: 4) instead of IDGVNHQHL (SEQ ID NO:3). When subjected to syfpeithi this peptide came out with a score of 29(25 for the native one). It is therefore likely that by substituting thefirst to amino-acids of this peptide by our cloning we have rendered itfrom cryptic to immunogenic. This is the first time that an epitope forthe HPV18E7 protein is described in the H-2D^(b) context.

Despite a high level of non specific background, these results also showthat, CyaA-HPV16+18E7_(Full) and CyaA-HPV16+18E7_(Δ) are able to deliverin vivo the H-2D^(b)-restricted T-cell epitope of the HPV16-E7 proteininto the cytosol of immunocompetent cells for processing andpresentation into the MHC class I pathway to elicit strong CTLresponses. CyaA-HPV16+18E7_(Δ) display a greater ability to do so ascompared to that of CyaA-HPV16+18E7_(Full). These data also show for thefirst time that CyaA carrying large polypeptidic fragments (up to 203amino acids) is still immunogenic.

A further experiment was done in order to confirm that described in FIG.16. The two remaining mice in each group were treated separately fromthe first ones. The experimental set-up is similar. The conclusions areidentical to those in FIG. 16 except that the high level of non specificlysis of EL4 cells by CTLs from CyaAE5-HPV18E7_(Δ32-42) vaccinated miceis likely to be due to an excess of HPV18E7₄₁₋₄₉ peptide remaining inthe flasks.

There is also a lysis of TC-1 cells with splenocytes from micevaccinated with CyaA-HPV16+18ΔE7 but not with CyaA-HPV16+18E7 this time.In this experiment, the non-specific background peaks at 40%.Nevertheless the conclusions that can be drawn from FIG. 17 confirmthose drawn from FIG. 16, that is:

-   -   CyaA-HPV18E7_(Δ32-42) is able to deliver in vivo a computer        predicted CD8⁺ H-2D^(b)-restricted T-cell epitope of the        HPV18-E7 protein into the cytosol of immunocompetent cells for        processing and presentation into the MHC class I pathway, to        elicit strong CTL responses. This is the first time that an        epitope for the HPV18E7 protein is described in the H-2D^(b)        context.    -   Recombinant CyaA carrying large polypeptidic fragments (up to        203 amino acids) is still immunogenic.

Conclusion

It was decided to construct a bivalent therapeutic vaccine carrying E7from HPV16 and HPV18. Two possible strategies have been considered; (i)the first one to mix equimolar quantities of two recombinant CyaAscarrying E7 from HPV16 on one hand and E7 from HPV18 on the other; (ii)the second one is to construct a recombinant CyaA carrying both E7proteins from HPV16 and HPV18. The present results describe theconstruction of recombinant CyaAs carrying E7 from HPV18 or E7 from bothviruses. The immunogenicity of the constructions were tested in a CTLassay.

The main conclusion from this study is that both considered strategiesare feasible since, recombinant CyaA carrying HPV18E7 sub-fragments isfunctional as it revealed for the first time a crypticH-2D^(b)-restricted epitope within the sequence of HPV18E7. Moreoverrecombinant CyaAs carrying E7 proteins (or sub-fragments) from bothHPV16 and 18 viruses were still and most interestingly immunogenic asthey were able to prime CTL response against H-2D^(b)-restricted HPV16E7epitopes naturally presented by TC-1 cells.

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The invention claimed is:
 1. A therapeutic vaccine composition,comprising at least one chimeric recombinant Bordetella sp. adenylatecyclase (CyaA) protein or fragment thereof, the CyaA protein or fragmentthereof comprising at least one inserted human papilloma virus (HPV) E7epitope, wherein administering the therapeutic vaccine composition to amammalian subject infected with HPV prevents HPV-induced tumor growth inthe subject.
 2. The therapeutic vaccine composition of claim 1, whereinadministering the therapeutic vaccine composition to the mammaliansubject reduces HPV induced tumor growth in the subject.
 3. Thetherapeutic vaccine composition of claim 1, wherein administering thetherapeutic vaccine composition to the mammalian subject causesregression of an HPV induced tumor in the subject.
 4. The therapeuticvaccine composition of claim 1, wherein administering the therapeuticvaccine composition to the mammalian subject protects against the onsetof malignant transformation by HPV in the subject.
 5. The therapeuticvaccine composition of claim 1, wherein the chimeric recombinant CyaAprotein or fragment thereof retains the property of the CyaA protein totarget CD11b/CD18 Antigen Presenting Cells (APC).
 6. The therapeuticvaccine composition of claim 1, wherein the chimeric recombinant CyaAprotein or fragment thereof retains the property of the CyaA protein tocause translocation of the at least one inserted HPV E7 epitope into thecytosol of targeted cells in the subject.
 7. The therapeutic vaccinecomposition of claim 1, wherein the at least one inserted HPV E7 epitopeis from an oncogenic HPV selected from oncogenic HPV16, HPV18, HPV31,HPV33, HPV35, HPV45, HPV52, and HPV58.
 8. The therapeutic vaccinecomposition of claim 1, wherein the at least one inserted HPV E7 epitopeis from HPV16.
 9. The therapeutic vaccine composition of claim 1,wherein the at least one inserted HPV E7 epitope is from HPV18.
 10. Thetherapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein or fragment thereof comprises from about 30 toabout 1300 amino acid residues of the CyaA protein, wherein the about 30to about 1300 amino acid residues comprise at least one fragmentselected from amino acid residues 1208 to 1243 of the CyaA protein ofBordetella pertussis and amino acid residues 1188 to 1281 of the CyaAprotein of Bordetella pertussis.
 11. The therapeutic vaccine compositionof claim 1, wherein the chimeric recombinant CyaA protein or fragmentthereof does not comprise amino acid residues 225 to 234 of the CyaAprotein.
 12. The therapeutic vaccine composition of claim 1, wherein thechimeric recombinant CyaA protein or fragment thereof comprises aninserted polypeptide comprising from about 5 to about 500 amino acidresidues that comprises the at least one inserted HPV E7 epitope. 13.The therapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein or fragment thereof comprises an insertedpolypeptide comprising from about 5 to about 200 amino acid residuesthat comprises the at least one inserted HPV E7 epitope.
 14. Thetherapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein or fragment thereof comprises an insertedpolypeptide comprising from about 10 to about 50 amino acid residuesthat comprises the at least one inserted HPV E7 epitope.
 15. Thetherapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein or fragment thereof is obtained by insertion ofat least two fragments of the native E7 protein in at least twopermissive sites of the CyaA protein or fragment thereof, wherein theorientation of the at least two inserted fragments is reversed withrespect to their natural location in the E7 protein.
 16. The therapeuticvaccine composition of claim 1, wherein the chimeric recombinant CyaAprotein or fragment thereof comprises an inserted polypeptide comprisingresidues 1 to 29 of the E7 protein of HPV16, or comprising residues 43to 98 of E7 protein of HPV16, or both fragments inserted in differentpermissive sites.
 17. The therapeutic vaccine composition of claim 1,wherein the chimeric recombinant CyaA protein or fragment thereofcomprises an inserted polypeptide comprising at least one sequenceselected from amino acid sequence RAHYNIVTF (SEQ ID NO: 1) (E7₄₉₋₅₇) andamino acid sequence GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 2)(E7₄₃₋₇₇).
 18. The therapeutic vaccine composition of claim 1, whereinthe CyaA protein or fragment thereof is the Bordetella pertussis CyaAprotein or a fragment thereof.
 19. The therapeutic vaccine compositionof claim 1, wherein the enzymatic activity of the CyaA protein orfragment thereof has been inactivated.
 20. The therapeutic vaccinecomposition of claim 19, wherein the enzymatic activity of the CyaAprotein or fragment thereof has been genetically inactivated.
 21. Thetherapeutic vaccine composition of claim 20, wherein the enzymaticactivity of the CyaA protein or fragment thereof has been geneticallyinactivated as a result of a dipeptide inserted in a site of the aminoacid sequence of CyaA involved in cyclase activity.
 22. The therapeuticvaccine composition of claim 21, wherein the dipeptide is inserted intothe CyaA protein or fragment thereof between residues 188 and 189 of thenative CyaA protein.
 23. The therapeutic vaccine composition of claim 1,wherein the chimeric recombinant CyaA protein is encoded by the insertcontained in plasmid pTRACE5-HPV16E7_(FULL) (C.N.C.M. 1-3191).
 24. Thetherapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein is encoded by the insert contained in plasmidpTRACE5-HPV16E7_(Δ30) _(—) ₄₂ (C.N.C.M. 1-3190).
 25. The therapeuticvaccine composition of claim 1, wherein the vaccine composition elicitsa cell-mediated immune response when administered to a mammaliansubject.
 26. The therapeutic vaccine composition of claim 1, wherein thevaccine composition elicits a humoral immune response when administeredto a mammalian subject.
 27. The therapeutic vaccine composition of claim1, further comprising at least one of a physiologically acceptablevehicle, a physiologically acceptable excipient, a physiologicallyacceptable carrier, and a physiologically acceptable diluent.
 28. Thetherapeutic vaccine composition of claim 1, further comprising at leastone of an adjuvant, a surfactant, and an immunomodulating substance. 29.The therapeutic vaccine composition of claim 1, wherein the chimericrecombinant CyaA protein or fragment thereof comprises at least two E7epitopes inserted in different permissive sites.
 30. The therapeuticvaccine composition of claim 29, wherein the at least two inserted E7epitopes are a fragment of the E7 protein of HPV 16 and a fragment ofthe E7 protein of HPV18.
 31. The therapeutic vaccine composition ofclaim 29, wherein the at least two inserted E7 epitopes are fragments ofthe E7 protein of HPV
 16. 32. The therapeutic vaccine composition ofclaim 29, wherein the at least two inserted E7 epitopes are fragments ofthe E7 protein of HPV18.
 33. The therapeutic vaccine composition ofclaim 31, wherein at least one of the fragments of the E7 protein ofHPV16 is selected from a fragment comprising the first 29 amino acidresidues of HPV16-E7 inserted into the CyaA protein or fragment thereofbetween residues 319 and 320 of the native CyaA protein and a fragmentcomprising amino acid residues 43 to 98 of HPV16-E7 inserted into theCyaA protein or fragment thereof between residues 224 and 235 of thenative CyaA protein.
 34. The therapeutic vaccine composition of claim33, wherein the chimeric recombinant CyaA protein or fragment thereofcomprises the fragment comprising the first 29 amino acid residues ofHPV16-E7 inserted between residues 319 and 320 of the native CyaAprotein and the fragment comprising amino acid residues 43 to 98 ofHPV16-E7 inserted between residues 224 and 235 of the native CyaAprotein.
 35. The therapeutic vaccine composition of claim 32, wherein atleast one of the fragments of the E7 protein of HPV18 is selected from afragment comprising the first 31 amino acid residues of HPV18-E7inserted into the CyaA protein or fragment thereof between residues 319and 320 of the native CyaA protein and a fragment comprising amino acidresidues 43 to 105 of HPV 18-E7 inserted into the CyaA protein orfragment thereof between residues 224 and 235 of the native CyaAprotein.
 36. The therapeutic vaccine composition of claim 35, whereinthe CyaA protein or fragment thereof comprises the fragment comprisingthe first 31 amino acid residues of HPV18-E7 inserted between residues319 and 320 of the native CyaA protein and the fragment comprising aminoacid residues 43 to 105 of HPV 18-E7 inserted between residues 224 and235 of the native CyaA protein.
 37. The therapeutic vaccine compositionof claim 1, comprising at least two different chimeric recombinant CyaAproteins or fragments thereof.
 38. The therapeutic vaccine compositionof claim 37, wherein the first of the at least two chimeric recombinantCyaA proteins or fragments thereof comprises a fragment of the E7protein of HPV16 selected from a fragment comprising the first 29 aminoacid residues of HPV16-E7 inserted between residues 319 and 320 of thenative CyaA protein and a fragment comprising amino acid residues 43 to98 of HPV16-E7 inserted between residues 224 and 235 of the native CyaAprotein; and wherein the second of the at least two chimeric recombinantCyaA proteins or fragments thereof comprises a fragment of the E7protein of HPV18 selected from a fragment comprising the first 31 aminoacid residues of HPV18-E7 inserted between residues 319 and 320 of thenative CyaA protein and a fragment comprising amino acid residues 43 to105 of HPV 18-E7 inserted between residues 224 and 235 of the nativeCyaA protein.
 39. The therapeutic vaccine composition of claim 38,wherein the first of the at least two CyaA proteins or fragments thereofcomprises the fragment comprising the first 29 amino acid residues ofHPV16-E7 inserted between residues 319 and 320 of the native CyaAprotein and comprises the fragment comprising amino acid residues 43 to98 of HPV16-E7 inserted between residues 224 and 235 of the native CyaAprotein; and wherein the second of the at least two CyaA proteins orfragments thereof comprises the fragment comprising first 31 amino acidresidues of HPV18-E7 inserted between residues 319 and 320 of the nativeCyaA protein and the fragment comprising amino acid residues 43 to 105of HPV 18-E7 inserted between residues 224 and 235 of the native CyaAprotein.
 40. The therapeutic vaccine composition of claim 29, whereinthe chimeric recombinant CyaA protein or fragment thereof comprisesresidues 43 to 98 of HPV16-E7 followed by residues 43 to 105 of HPV18-E7inserted between residues 224 and 235 of the native CyaA protein and thefirst 31 amino acid residues of HPV18-E7 followed by the first 29 aminoacid residues of HPV16-E7 inserted between residues 319 and 320 of thenative CyaA protein.
 41. The therapeutic vaccine composition of claim22, wherein the CyaA protein or fragment thereof is enzymaticallyinactivated by insertion of a LQ dipeptide between amino acid residuesAsp 188 and Ile 189 of the native CyaA protein.
 42. The therapeuticvaccine composition of claim 1, wherein the at least one inserted HPV E7epitope has been modified with respect to its native amino acid sequenceby addition of non-naturally flanking sequences.
 43. The therapeuticvaccine composition of claim 1, wherein the subject is a human.